Pan-genotypic agents against influenza virus and methods of using the same

ABSTRACT

Methods of inhibiting influenza A virus in a sample are provided. Aspects of the methods include contacting a sample comprising viral RNA (vRNA) having a PSL2 motif with an effective amount of an agent that specifically binds the PSL2 motif to inhibit the influenza A virus. Also provided are methods of treating or preventing influenza A virus infection in a subject. Also provided are methods for screening a candidate agent for the ability to inhibit influenza A virus in a cell, the method comprising: contacting a sample with a candidate agent; and determining whether the candidate agent specifically binds to the PSL2 motif of vRNA. Also provided are compounds and pharmaceutical compositions comprising an oligonucleotide sequence complementary to a PB2 vRNA region that find use in the subject methods.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional PatentApplication No. 62/302,548, filed Mar. 2, 2016, which application isincorporated herein by reference in its entirety.

INTRODUCTION

Influenza A virus (IAV) is a segmented RNA virus that causes significantmorbidity and mortality worldwide. All currently approved IAV antiviraldrugs are targeted against viral proteins, are subtype limited, and arechallenged by rising antiviral resistance against all drug classmembers.

The IAV genome consists of eight single-stranded negative-sense viralRNA (vRNA) segments that encode a minimum of 14 known viral proteins.The vRNA, together with nucleoprotein (NP) and the heterotrimericpolymerase complex, comprised of PB2, PB1, and PA proteins, forms thecomplete viral ribonucleoprotein (vRNP). To be fully infectious, IAVvirions must incorporate at least one of each segment's vRNP. Each vRNPinteracts with at least one other partner to form a supramolecularcomplex likely maintained by intersegment RNA-RNA and/or protein-RNAinteractions hypothesized to guide the packaging process.

SUMMARY

Aspects of the present disclosure provide pan-genotypic compositionsdesigned to disrupt an RNA structural element of IAV, called PackagingStem-Loop 2 (PSL2), within the 5′ packaging signal region of genomesegment PB2. Disruption of PSL2 structure dramatically inhibits IAV.PSL2 is conserved across all tested influenza A subtypes.

Methods of inhibiting influenza A virus in a sample are provided.Aspects of the methods include contacting a sample comprising viral RNA(vRNA) having a PSL2 motif with an effective amount of an agent thatspecifically binds the PSL2 motif to inhibit the influenza A virus. Insome cases, the vRNA is isolated from a virion or a cell. In some cases,the vRNA is in a virion. In some cases, the vRNA is in an infected cell.Also provided are methods of treating or preventing influenza A virusinfection in a subject. Also provided are methods for screening acandidate agent for the ability to inhibit influenza A virus in a cell,the method comprising: contacting a sample with a candidate agent; anddetermining whether the candidate agent specifically binds to the PSL2motif of vRNA. Also provided are compounds and pharmaceuticalcompositions comprising an oligonucleotide sequence complementary to aPB2 vRNA region that find use in the subject methods.

BRIEF DESCRIPTION OF THE FIGURES

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIG. 1A-FIG. 1F depicts RNA secondary structures of wild-type PB2 (SEQID NO: 1) and packaging mutant vRNAs, PB2m757 (SEQ ID NO: 2), m745 (SEQID NO: 3), 1918 pandemic (H1N1) (SEQ ID NO: 4), High-path avian (H5M=N1)(SEQ ID NO: 5) and 2009 swine (H1N1) (SEQ ID NO: 6).

FIG. 2, panels A and B, depicts the reactivity of full-length PB2 vRNA.

FIG. 3A-FIG. 3E depicts the disruption of wild-type reactivity (SEQ IDNO: 7) by packaging-defective mutations, PB2m744b (SEQ ID NO: 8),PB2m745 (SEQ ID NO: 9), PB2m55c (SEQ ID NO: 10) and PB2m757 (SEQ ID NO:11).

FIG. 4 depicts the conservancy of nucleotide sequence containing thePSL2 structure.

FIG. 5A-FIG. 5D depicts the 2-dimensional Mutate-and-Map analysis ofPSL2 RNA secondary structure (FIG. 5C, SEQ ID NO: 12).

FIG. 6, panels A-B, depicts the design of compensatory mutations topreviously described PR8 PB2 mutants (panel A, SEQ ID NO: 13).

FIG. 7, panels A-D, depicts synonymous mutation of single highlyconserved codons of the PR8 PB2 vRNA (panel A, SEQ ID NOs: 14-15; panelB, SEQ ID NOs: 16-23 top to bottom).

FIG. 8 depicts a table showing PB2 packaging mutant nomenclature andcorresponding sites of mutation.

FIG. 9A-FIG. 9C depicts the effect of synonymous mutation on PSL2structure, PB2m731 (SEQ ID NO: 24), PB2m751 (SEQ ID NO: 25) and PB2m748(SEQ ID NO: 26).

FIG. 10, panels A-I, depicts the effect of compensatory mutations in PR8PB2 packaging-defective mutants on viral packaging and titer.

FIG. 11 panels A-D, depicts multidimensional chemical mapping of PB2packaging-defective and compensatory mutant partners (panel B, SEQ IDNO: 27).

FIG. 12, panels a-t, depicts 2-dimensional Mutate-Map-Rescue analysis.

FIG. 13 depicts the design of primer sequences for 2-dimensionalMutate-Map-Rescue mutants. Sequences correspond to SEQ ID NOs: 28-43,top to bottom.

FIG. 14, panels A-B, depicts that packaging-defective viruses areattenuated in vivo.

FIG. 15, panels A-D, depicts antiviral activity of Locked Nucleic Acidstargeting PSL2 RNA structure (panel A, SEQ ID NO: 44).

FIG. 16A-FIG. 16B depicts analysis on LNA-RNA binding.

FIG. 17, panels A-B shows the percent survival and weight loss of miceover time after intranasal administration of a single dose of exemplarycompound LNA9.

FIG. 18, panels A-D, show the susceptibility of influenza virus tooseltamivir after serial passages under drug pressure.

FIG. 19, panels A-C, show the susceptibility of influenza virus toexemplary compound LNA9, including viruses after serial passages underdrug pressure and drug resistant virus.

DEFINITIONS

Before describing exemplary embodiments in greater detail, the followingdefinitions are set forth to illustrate and define the meaning and scopeof the terms used in the description.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton, et al., DICTIONARYOF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, NewYork (1994), and Hale & Markham, THE HARPER COLLINS DICTIONARY OFBIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with thegeneral meaning of many of the terms used herein. Still, certain termsare defined below for the sake of clarity and ease of reference.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. For example, the term “a primer”refers to one or more primers, i.e., a single primer and multipleprimers. It is further noted that the claims can be drafted to excludeany optional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely,”“only” and the like in connection with the recitation of claim elements,or use of a “negative” limitation.

The term “sample” as used herein relates to a material or mixture ofmaterials, typically, although not necessarily, in fluid form,containing one or more components of interest.

As used herein, the term “effective amount” refers to that amount of asubstance (e.g., an agent of interest) that produces some desired localor systemic effect. Effective amounts of active agents of interest varydepending on a variety of factors including, but not limited to, theweight and age of the subject, the condition being treated, the severityof the condition, the manner of administration and the like, and canreadily be determined, e.g., determined empirically using data such asthat data provided in the experimental section below.

The term “sample” as used herein relates to a material or mixture ofmaterials, typically, although not necessarily, in fluid, i.e., aqueous,form, containing one or more components of interest. Samples may bederived from a variety of sources such as from a biological sample orsolid, such as tissue or fluid isolated from an individual, includingbut not limited to, for example, plasma, serum, spinal fluid, semen,lymph fluid, the external sections of the skin, respiratory, intestinal,and genitourinary tracts, tears, saliva, milk, blood cells, tumors,organs, and also samples of in vitro cell culture constituents(including but not limited to conditioned medium resulting from thegrowth of cells in cell culture medium, putatively virally infectedcells, recombinant cells, and cell components). Components in a sampleare termed “analytes” herein. In many embodiments, the sample is acomplex sample containing at least about 10², 5×10², 10³, 5×10³, 10⁴,5×10⁴, 10⁵, 5×10⁵, 10⁶, 5×10⁶, 10⁷, 5×10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹² ormore species of analyte.

“Antibody fragments” comprise a portion of an intact antibody, forexample, the antigen binding or variable region of the intact antibody.Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fvfragments; diabodies; linear antibodies (Zapata et al., Protein Eng.8(10): 1057-1062 (1995)); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments. Papaindigestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, a designation reflecting the abilityto crystallize readily. Pepsin treatment yields an F(ab′)2 fragment thathas two antigen combining sites and is still capable of cross-linkingantigen.

The terms “polypeptide” and “protein”, used interchangeably herein,refer to a polymeric form of amino acids of any length, which caninclude coded and non-coded amino acids, chemically or biochemicallymodified or derivatized amino acids, and polypeptides having modifiedpeptide backbones. The term “fusion protein” or grammatical equivalentsthereof is meant a protein composed of a plurality of polypeptidecomponents, that while typically unjoined in their native state,typically are joined by their respective amino and carboxyl terminithrough a peptide linkage to form a single continuous polypeptide.Fusion proteins may be a combination of two, three or even four or moredifferent proteins. The term polypeptide includes fusion proteins,including, but not limited to, fusion proteins with a heterologous aminoacid sequence, fusions with heterologous and homologous leadersequences, with or without N-terminal methionine residues;immunologically tagged proteins; fusion proteins with detectable fusionpartners, e.g., fusion proteins including as a fusion partner afluorescent protein, β-galactosidase, luciferase, etc.; and the like.

In general, polypeptides may be of any length, e.g., greater than 2amino acids, greater than 4 amino acids, greater than about 10 aminoacids, greater than about 20 amino acids, greater than about 50 aminoacids, greater than about 100 amino acids, greater than about 300 aminoacids, usually up to about 500 or 1000 or more amino acids. “Peptides”are generally greater than 2 amino acids, greater than 4 amino acids,greater than about 10 amino acids, greater than about 20 amino acids,usually up to about 50 amino acids. In some embodiments, peptides arebetween 5 and 30 amino acids in length.

The term “specific binding” refers to the ability of an agent topreferentially bind to a particular target (e.g., PSL2) that is presentin a homogeneous mixture of different analytes. In some cases, aspecific binding interaction will discriminate between desirable andundesirable analytes in a sample, typically more than about 10 to100-fold or more (e.g., more than about 1000-fold). In some cases, theaffinity between an agent of interest and analyte (e.g., PSL2) when theyare specifically bound in a capture agent/analyte complex is at least10⁻⁸M, at least 10⁻⁹M, usually up to about 10⁻¹⁰ M. Specific binding caninclude hybridization, polypeptide-nucleic acid interactions or smallmolecule-nucleic acid interactions.

“Oligonucleotide” refers to ribose and/or deoxyribose nucleoside subunitpolymers having between about 2 and about 200 contiguous subunits. Thenucleoside subunits can be joined by a variety of intersubunit linkages,including, but not limited to, phosphodiester, phosphotriester,methylphosphonate, P3′→N5′ phosphoramidate, N3′→P5′ phosphoramidate,N3′→P5′ thiophosphoramidate, and phosphorothioate linkages. Further,“oligonucleotides” includes modifications, known to one skilled in theart, to the sugar (e.g., 2′ substitutions), the base (see the definitionof “nucleoside” below), and the 3′ and 5′ termini. In embodiments wherethe oligonucleotide moiety includes a plurality of intersubunitlinkages, each linkage may be formed using the same chemistry or amixture of linkage chemistries may be used. The terms “oligonucleotide”,“nucleic acid,” “nucleic acid molecule,” “nucleic acid fragment,”“nucleic acid sequence or segment,” or “polynucleotide” are usedinterchangeably and may also be used interchangeably with gene, cDNA,DNA and RNA encoded by a gene.

A “Locked nucleic acid” (LNA) is a modified RNA nucleotide where theribose moiety is modified with an extra bridge connecting the 2′ oxygenand 4′ carbon. The bridge “locks” the ribose in the 3′-endoconformation, which is often found in the A-form duplexes. LNAnucleotides can be mixed with any convenient nucleotides or nucleotideanalogs, such as DNA or RNA residues in an oligonucleotide wheneverdesired. LNA's hybridize with DNA or RNA according to Watson-Crickbase-pairing rules. Such oligomers can be synthesized chemically. Ingeneral, the locked ribose conformation enhances base stacking andbackbone pre-organization to increase the hybridization properties(melting temperature) of the oligonucleotide.

The disclosure encompasses isolated or substantially purified nucleicacid nucleic acid molecules and compositions containing those molecules.In the context of the present disclosure, an “isolated” or “purified”DNA molecule or RNA molecule is a DNA molecule or RNA molecule thatexists apart from its native environment and is therefore not a productof nature. An isolated DNA molecule or RNA molecule may exist in apurified form or may exist in a non-native environment such as, forexample, a transgenic host cell. For example, an “isolated” or“purified” nucleic acid molecule or biologically active portion thereof,is substantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized. In oneembodiment, an “isolated” nucleic acid is free of sequences thatnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. For example, in various embodiments,the isolated nucleic acid molecule can contain less than about 5 kb, 4kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences thatnaturally flank the nucleic acid molecule in genomic DNA of the cellfrom which the nucleic acid is derived. Fragments and variants of thedisclosed nucleotide sequences are also encompassed by the presentdisclosure. By “fragment” or “portion” is meant a full length or lessthan full length of the nucleotide sequence. The siRNAs of the presentdisclosure can be generated by any method known to the art, for example,by in vitro transcription, recombinantly, or by synthetic means. In oneexample, the siRNAs can be generated in vitro by using a recombinantenzyme, such as T7 RNA polymerase, and DNA oligonucleotide templates.

A “small interfering” or “short interfering RNA” or siRNA is a RNAduplex of nucleotides that is targeted to a gene interest. A “RNAduplex” refers to the structure formed by the complementary pairingbetween two regions of a RNA molecule. siRNA is “targeted” to a gene inthat the nucleotide sequence of the duplex portion of the siRNA iscomplementary to a nucleotide sequence of the targeted gene. In someembodiments, the length of the duplex of siRNAs is less than 30nucleotides. In some embodiments, the duplex can be 29, 28, 27, 26, 25,24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 nucleotidesin length. In some embodiments, the length of the duplex is 19-25nucleotides in length. The RNA duplex portion of the siRNA can be partof a hairpin structure. In addition to the duplex portion, the hairpinstructure may contain a loop portion positioned between the twosequences that form the duplex. The loop can vary in length. In someembodiments the loop is 5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides inlength. The hairpin structure can also contain 3′ or 5′ overhangportions. In some embodiments, the overhang is a 3′ or a 5′ overhang 0,1, 2, 3, 4 or 5 nucleotides in length.

The term “lipid” is used broadly herein to encompass substances that aresoluble in organic solvents, but sparingly soluble, if at all, in water.The term lipid includes, but is not limited to, hydrocarbons, oils, fats(such as fatty acids, glycerides), sterols, steroids and derivativeforms of these compounds. Preferred lipids are fatty acids and theirderivatives, hydrocarbons and their derivatives, and sterols, such ascholesterol. As used herein, the term lipid also includes amphipathiccompounds which contain both lipid and hydrophilic moieties. Fatty acidsusually contain even numbers of carbon atoms in a straight chain(commonly 12-24 carbons) and may be saturated or unsaturated, and cancontain, or be modified to contain, a variety of substituent groups. Forsimplicity, the term “fatty add” also encompasses fatty acidderivatives, such as fatty amides produced by the conjugation reactions,e.g., with a modified terminal of an oligonucleotide.

Other definitions of terms may appear throughout the specification.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

Before the various embodiments are described, it is to be understoodthat the teachings of this disclosure are not limited to the particularembodiments described, and as such can, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present teachings will be limited onlyby the appended claims.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described inany way. While the present teachings are described in conjunction withvarious embodiments, it is not intended that the present teachings belimited to such embodiments. On the contrary, the present teachingsencompass various alternatives, modifications, and equivalents, as willbe appreciated by those of skill in the art.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present teachings, some exemplarymethods and materials are now described.

The citation of any publication is for its disclosure prior to thefiling date and should not be construed as an admission that the presentclaims are not entitled to antedate such publication by virtue of priorinvention. Further, the dates of publication provided can be differentfrom the actual publication dates which can be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which can be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentteachings. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

All patents and publications, including all sequences disclosed withinsuch patents and publications, referred to herein are expresslyincorporated by reference.

Methods for Inhibiting Influenza a Virus

As summarized above, aspects of the present disclosure includepan-genotypic compositions designed to disrupt an RNA structural elementof IAV, called Packaging Stem-Loop 2 (PSL2), within the 5′ packagingsignal region of genome segment PB2. By “pan-genotypic” is meant thecompositions are effective across a variety of different types of IAVwhere the PSL2 structural element is conserved. In some cases, thesubject compositions can be referred to as broad spectrum. As usedherein, the term “broad spectrum” refers to the anti-viral activity of asingle moiety that is active against two or more different viruses, suchas three or more, four or more, five or more, six or more, eight ormore, 10 or more different viruses. The two or more different virusesmay be selected from different virus sub-groups (e.g., Influenza A group1 or Influenza A group 2), or may be selected from within the same group(e.g., two or more of H1, H2, H5, H6, H8 and H9 group 1 influenza Aviruses, or two or more of H3, H4, H7 and H10 Group 2 Influenza Aviruses).

Disruption of PSL2 structure dramatically inhibits IAV. PSL2 isconserved across all tested influenza A subtypes. FIG. 1, panel a, showsan example of the PSL2 structure that can be targeted in the subjectmethods. FIG. 4 illustrates the conservancy of nucleotide sequences ofinterest containing the PSL2 structure. In some cases, the subjectcompositions have broad spectrum activity against IAVs, such as activityagainst 2 or more IAVs selected from H1N1, H3N2 and HSN.

Aspects of the present disclosure include methods for inhibitinginfluenza A virus (IAV) in a sample. In some embodiments, the methodincludes contacting a sample comprising viral RNA (vRNA) having a PSL2motif with an effective amount of an agent that specifically binds thePSL2 motif to inhibit the influenza A virus. In some cases, the sampleis in vitro. In certain cases, the sample is in vivo. The vRNA in thesample can be comprised in a virion. In some cases, the vRNA iscomprised in a cell, such as a cell infected with the virus particle.

Aspects of the present disclosure include methods for inhibitinginfluenza A virus (IAV) in a cell. In some embodiments, the methodincludes contacting a cell comprising viral RNA (vRNA) having a PSL2motif with an effective amount of an agent that specifically binds thePSL2 motif to inhibit the influenza A virus. In some cases, the cell isin vitro. In certain cases, the cell is in vivo.

In some embodiments, the vRNA in the sample (e.g., cell) comprises PB2vRNA. As used here, by “PB2 vRNA” is meant viral RNA (e.g., IAV RNA)that includes the conserved RNA structural element called PackagingStem-Loop 2 (PSL2). The agent can bind to particular sites of the PSL2motif to disrupt the overall structure of the vRNA thereby inhibitingthe virus (see e.g., FIG. 14). For example, FIG. 1 illustrates RNAsecondary structures of wild-type PB2 and packaging mutant vRNAs.

In some embodiments, contacting the sample (e.g., cell) with an agentresults in at least 2 log₁₀ titer deficits of the virus, such as atleast 2.5, at least 3, at least 3.5, at least 4, at least 5, at least 6,at least 7, at least 8, at least 9, at least 10 log₁₀ titer deficits ofthe virus. In some embodiments, the agent is an oligonucleotide compound(e.g., as described herein) comprising a sequence complementary to aPSL2 motif of the vRNA, or a salt thereof.

In some instances of the methods, binding (e.g., via hybridization) ofthe oligonucleotide compound (e.g., one of the sequences describedabove) to the region of PB2 vRNA disrupts the overall secondary RNAstructure of the PB2 vRNA. In some cases, the subject compound targetsat least part of the region defined by nucleotides 34-87 in the(−)-sense notation of the 5′ terminal coding region of the PB2 vRNA. Insome cases, the compound targets at least part of the region defined bynucleotides 1-14 in the (−)-sense notation of the 5′ terminal codingregion of the PB2 vRNA. In some instances of the methods, the methodfurther includes recruiting an RNase to the PSL2 to degrade the vRNA.

Aspects of the present disclosure include a method of treating orpreventing influenza A virus infection in a subject. In someembodiments, the method comprises administering to a subject in needthereof a pharmaceutical composition comprising an effective amount ofan active agent that specifically binds to a PSL2 motif of a viral RNA(vRNA) (e.g., as described herein). As such, in some cases, the subjectis one who has been infected with the virus. In certain cases, thesubject is one who is at risk of being infected, or is suspected ofbeing infected with the virus. In some embodiments, the vRNA is a PB2vRNA.

Any convenient protocol for administering the agent to a subject may beemployed. The particular protocol that is employed may vary, e.g.,depending on the site of administration and whether the agents are e.g.,oligonucleotides, antibodies, proteins, peptides or small molecules. Forin vivo protocols, any convenient administration protocol may beemployed. Depending upon the identity and binding affinity of the agent,the response desired, the manner of administration, e.g. locally orsystemic, intraocular, periocular, retrobalbar, intramuscular,intravenous, intraperitoneal, subcutaneous, subconjunctival, intranasal,topical, eye drops, i.v. s.c., i.p., oral, and the like, the half-life,the number of cells or size of the graft bed or transplanted tissue,various protocols may be employed.

Also provided are pharmaceutical compositions including the subjectagents. Any convenient excipients, carriers, etc. can be utilized in thecompositions. Pharmaceutically acceptable carriers that find use in thecompositions may include sterile aqueous of non-aqueous solutions,suspensions, and emulsions. Examples of non-aqueous solvents arepropylene glycol, polyethylene glycol, vegetable oils such as olive oil,and injectable organic esters such as ethyl oleate. Aqueous carriersinclude water, alcoholic/aqueous solutions, emulsions or suspensions,including saline and buffered media. Parenteral vehicles include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's or fixed oils. Intravenous vehicles include fluid andnutrient replenishers, electrolyte replenishers (such as those based onRinger's dextrose), and the like. Preservatives and other additives mayalso be present such as, for example, antimicrobials, antioxidants,chelating agents, and inert gases and the like. The agent compositionmay also be lyophilized, for subsequent reconstitution and use. Thecomposition can also include a carrier as described here. Examples ofcarriers which may be used include, but are not limited to, alum,microparticles, liposomes, and nanoparticles. Any convenient additivescan be included in the subject compositions to enhance the delivery ofthe subject active agent. Additives of interest include, cellular uptakeenhancers, carrier proteins, lipids, dendrimer carriers, carbohydrates,and the like.

In some cases, the pharmaceutical composition further includes one ormore additional active agents. Active agents of interest include anadditional oligonucleotide compound of the present disclosure and anyconvenient antiviral compounds or drugs of interest. including but notlimited to Amantadine, Rimantadine, Zanamivir, Oseltamivir, Peramivirand the like.

Agents

Any convenient agents may be utilized as an agent of a target ofinterest (e.g., PSL2) in the subject methods and compositions. Agents ofinterest include, but are not limited to, a ligand of PSL-2, aPSL2-binding antibody, a scaffolded protein binder of PSL2, anoligonucleotide, a small molecule, and a peptide; or a fragment,variant, or derivative thereof; or combinations of any of the foregoing.

Antibodies that may be used as agents in connection with the presentdisclosure can encompass, but are not limited to, monoclonal antibodies,polyclonal antibodies, bispecific antibodies, Fab antibody fragments,F(ab)₂ antibody fragments, Fv antibody fragments (e.g., V_(H) or V_(L)),single chain Fv antibody fragments and dsFv antibody fragments.Furthermore, the antibody molecules may be fully human antibodies,humanized antibodies, or chimeric antibodies. The antibodies that may beused in connection with the present disclosure can include any antibodyvariable region, mature or unprocessed, linked to any immunoglobulinconstant region. Minor variations in the amino acid sequences ofantibodies or immunoglobulin molecules are encompassed by the presentdisclosure, providing that the variations in the amino acid sequencemaintain 75% or more, e.g., 80% or more, 90% or more, 95% or more, or99% or more of the sequence. In particular, conservative amino acidreplacements are contemplated. Conservative replacements are those thattake place within a family of amino acids that are related in their sidechains. Whether an amino acid change results in a functional peptide canbe determined by assaying the specific activity of the polypeptidederivative.

In some embodiments, the agent is an antibody fragment (e.g., asdescribed herein).

In some embodiments, the agent is a scaffolded polypeptide binder. Ascaffold refers to an underlying peptidic framework (e.g., a consensussequence or structural motif) from which a polypeptide agent arose. Theunderlying scaffold sequence includes those residues that are fixed andvariant residues that may confer on the resulting polypeptide agentsdifferent functions, such as specific binding to a target receptor. Suchstructural motifs may be characterized and compared structurally as acombination of particular secondary and tertiary structural elements, oralternatively, as a comparable primary sequence of amino acid residues.Any convenient scaffolds and scaffolded polypeptides may be utilized asagents in the subject methods. In some embodiments, such agents may beidentified utilizing a recombinant screening method such as phagedisplay screening. Scaffolded polypeptide binders of interest include,but are not limited to, synthetic small proteins and recombinant smallproteins such as Affibodies.

In some cases, the agent is a small molecule that binds PSL2. Smallmolecules of interest include, but are not limited to, small organic orinorganic compounds having a molecular weight (MW) of more than 50 andless than about 2,500 daltons (Da), such as more than 50 and less thanabout 1000 Da, or more than 50 and less than about 500 Da. “Smallmolecules” encompasses numerous biological and chemical classes,including synthetic, semi-synthetic, or naturally-occurring inorganic ororganic molecules, including synthetic, recombinant ornaturally-occurring polypeptides and nucleic acids. Small molecules ofinterest can comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and caninclude at least an amine, carbonyl, hydroxyl or carboxyl group, and cancontain at least two of the functional chemical groups. The smallmolecules can comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Small molecules are also found amongbiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof.

Oligonucleotide Compounds

In some embodiments, the agent is an oligonucleotide or derivativethereof, or a salt thereof (e.g., a pharmaceutically acceptable salt).In some instances, the oligonucleotide in complementary to a particularsegment of the PSL2 motif (e.g., as described herein). Complementaryoligonucleotides that find use in the subject methods will in some casesbe at least 5, such at least 6, at least 7 at least 8, at least 9, atleast 10, at least 11, about 12, at least 13, at least 14, at least 15,or even more. In some cases, the complementary oligonucleotide is 30nucleotides or less in length, such as 25 nucleotides or less in length,20 nucleotides or less in length, or 15 nucleotides or less in length,where the length is governed by efficiency of inhibition, specificity,including absence of cross-reactivity, and the like. The presentdisclosure provides for short oligonucleotides, e.g., of from 7 or 8 to15 nucleotides in length, can be strong and selective inhibitors of PSL2function. In some embodiments, the active agent is a compound comprisingan oligonucleotide sequence comprising at least 8 nucleoside subunitscomplementary to the region of PB2 vRNA. In some embodiments, the activeagent is a compound comprising an oligonucleotide sequence comprising atleast 8 and 20 or less (e.g., 15 or less) nucleoside subunitscomplementary to the region of PB2 vRNA.

A specific region or regions of the endogenous strand PSL2 sequence ischosen to be complemented by the oligonucleotide agent. Selection of aspecific sequence for the oligonucleotide may use an empirical method,where based on the structural analysis (e.g., as described herein)several candidate sequences are assayed for inhibition of the target IAVin an in vitro or animal model. A combination of oligonucleotides andsequences may also be used, where several regions of the target PSL2 areselected for antisense complementation.

In some embodiments, the agent is an oligonucleotide compound comprisingat least 5 nucleoside subunits (e.g., at least 6, at least 7, at least8, at least 9, at least 10, at least 12, at least 14, at least 16, atleast 18, or at least 20) complementary to a PSL2 motif of a vRNA, or asalt thereof. In certain embodiments, one or more of the linkages of theoligonucleotide are selected from methylphosphonate, P3′→N5′phosphoramidate, N3′→P5′ phosphoramidate, N3′→P5′ thiophosphoramidate,phosphorodithioate and phosphorothioate linkages. In certain instances,the oligonucleotide sequence is a locked nucleic acid. In certaininstances, the oligonucleotide sequence includes one or more lockednucleic acid nucleotides, such as 2 or more, 3 or more, 4 or more, 5 ormore, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or evenmore.

In some embodiments, the agent is an oligonucleotide that comprises atleast 5 deoxyribonucleotide units (e.g., units complementary to a PSL2motif) (e.g., least 6, at least 7, at least 8, at least 9, at least 10,at least 12, at least 14, at least 16, at least 18, at least 20) and iscapable of recruiting an RNase. In some case, the oligonucleotiderecruits an RNase to catalyze the degradation of the target vRNA intosmaller components. Any convenient methods and moieties for recruitingan RNase can be incorporated into the subject agents (e.g.,oligonucleotides). In some instances, the oligonucleotide agent furtherincludes a sequence that recruits an RNase of interest. Itis understoodthat unless indicated otherwise, an oligonucleotide sequence as depictedherein is meant to include DNA sequences, RNA sequences (e.g., where Ucan optionally replace T), mixed RNA/DNA sequences, and analogs thereof,including analogs where one or more nucleotides of the sequence aremodified nucleotides, such as IAA analogs, and/or analogs where one ormore internucleoside linkages are replaced, with a non-naturallyoccurring linkage such as a phosphorothioate, phosphorodithioate,phosphoramidate or thiophosphoramidate linkage.

In some embodiments, the oligonucleotide comprises a sequence selectedfrom:

(SEQ ID NO: 45) 5′ ACCAAAAGAAT 3′; (SEQ ID NO: 46) 5′ TGGCCATCAAT 3′;(SEQ ID NO: 47) 5′ TAGCATACTTA 3′; (SEQ ID NO: 48) 5′ CCAAAAGA 3′;(SEQ ID NO: 49) 5′ CATACTTA 3′; (SEQ ID NO: 50) 5′ CAGACACGACCAAAA 3′;(SEQ ID NO: 51) 5′ TACTTACTGACAGCC 3′; (SEQ ID NO: 52) 5′AGACACGACCAAAAG 3′; (SEQ ID NO: 53) 5′ ACCAAAAGAAT 3′; (SEQ ID NO: 54)5′ TGGCCATCAAT 3′; (SEQ ID NO: 55) 5′ TAGCATACTTA 3′; (SEQ ID NO: 56) 5′CGACCAAAAGAATTC 3′; (SEQ ID NO: 57) 5′ CGACCAAAAGAATTC 3′;(SEQ ID NO: 58) 5′ GATGGCCATCAATTA 3′; (SEQ ID NO: 59) 5′GATGGCCATCAATTA 3′; (SEQ ID NO: 60) 5′ TCTAGCATACTTACT 3′;(SEQ ID NO: 61) 5′ TCTAGCATACTTACT 3′; (SEQ ID NO: 62) 5′GAATTCGGATGGCCA 3′; (SEQ ID NO: 63) 5′ GGCCATCAATTAGTG 3′;(SEQ ID NO: 64) 5′ TTCGGATGGCCATCA 3′; (SEQ ID NO: 65) 5′AGCCAGACAGCGA 3′; and (SEQ ID NO: 66) 5′ GACAGCCAGACAGCA 3′.

In certain embodiments, the oligonucleotide comprises the sequence: 5′ACCAAAAGAAT 3′ (SEQ ID NO:45). In certain embodiments, theoligonucleotide comprises the sequence: 5′ TGGCCATCAAT 3′ (SEQ IDNO:46). In certain embodiments, the oligonucleotide comprises thesequence: 5′ TAGCATACTTA 3′ (SEQ ID NO:47). In certain embodiments, theoligonucleotide comprises the sequence: 5′ CCAAAAGA 3′ (SEQ ID NO:48).In certain embodiments, the oligonucleotide comprises the sequence: 5′CATACTTA 3′ (SEQ ID NO:49). In certain embodiments, the oligonucleotidecomprises the sequence: 5′ CAGACACGACCAAAA 3′ (SEQ ID NO:50). In certainembodiments, the oligonucleotide comprises the sequence: 5′TACTTACTGACAGCC 3′ (SEQ ID NO:51). In certain embodiments, theoligonucleotide comprises the sequence: 5′ AGACACGACCAAAAG 3′ (SEQ IDNO:52). In certain embodiments, the oligonucleotide comprises thesequence: 5′ ACCAAAAGAAT 3′ (SEQ ID NO:53). In certain embodiments, theoligonucleotide comprises the sequence: 5′ TGGCCATCAAT 3′ (SEQ IDNO:54). In certain embodiments, the oligonucleotide comprises thesequence: 5′ TAGCATACTTA 3′ (SEQ ID NO:55). In certain embodiments, theoligonucleotide comprises the sequence: 5′ CGACCAAAAGAATTC 3′ (SEQ IDNO:56). In certain embodiments, the oligonucleotide comprises thesequence: 5′ CGACCAAAAGAATTC 3′ (SEQ ID NO:57). In certain embodiments,the oligonucleotide comprises the sequence: 5′ GATGGCCATCAATTA 3′ (SEQID NO:58). In certain embodiments, the oligonucleotide comprises thesequence: 5′ GATGGCCATCAATTA 3′ (SEQ ID NO:59). In certain embodiments,the oligonucleotide comprises the sequence: 5′ TCTAGCATACTTACT 3′ (SEQID NO:60). In certain embodiments, the oligonucleotide comprises thesequence: 5′ TCTAGCATACTTACT 3′ (SEQ ID NO:61). In certain embodiments,the oligonucleotide comprises the sequence: 5′ GAATTCGGATGGCCA 3′ (SEQID NO:62). In certain embodiments, the oligonucleotide comprises thesequence: 5′ GGCCATCAATTAGTG 3′ (SEQ ID NO:63). In certain embodiments,the oligonucleotide comprises the sequence:5′ TTCGGATGGCCATCA 3′ (SEQ IDNO:64). In certain embodiments, the oligonucleotide comprises thesequence: 5′ AGCCAGACAGCGA 3′ (SEQ ID NO:65). In certain embodiments,the oligonucleotide comprises the sequence: 5′ GACAGCCAGACAGCA 3′ (SEQID NO:66).

In some instances, binding (e.g., via hybridization) of theoligonucleotide compound (e.g., one of the sequences described above) tothe region of PB2 vRNA disrupts the overall secondary RNA structure ofthe PB2 vRNA.

The oligonucleotide sequences can include any convenient number of DNA,RNA and LNA nucleotides. In some instances of the oligonucleotidesequences described herein, the sequence is a mixed RNA/DNA sequence. Insome instances of the oligonucleotide sequences described herein, thesequence is a mixed LNA/DNA sequence. In some instances of theoligonucleotide sequences described herein, the sequence is a mixedLNA/RNA sequence. In some instances of the oligonucleotide sequencesdescribed herein, the sequence includes only LNA nucleotides. In someinstances of the oligonucleotide sequences described herein, thesequence includes only DNA nucleotides. In some instances of theoligonucleotide sequences described herein, the sequence includes onlyRNA nucleotides.

In certain instances, the subject oligonucleotide has one of thefollowing arrangements of types of nucleotide in the sequence (e.g., oneof SEQ ID NOs:45-66):

A, where A is a sequence of 8 or more LNA nucleotides;

A-B-A, where B is a sequence of 6-8 DNA nucleotides, and each A is asequence of 3-4 LNA nucleotides;

A-B-A, where B is a sequence of 7-8 DNA nucleotides, and each A is asequence of 4 LNA nucleotides;

L-D-L-D-L-D-L, where each L is a sequence of 1-2 LNA nucleotides, andeach D is a sequence of 2 DNA nucleotides;

L-D-L-D-L-D-L, where each L is a sequence of 1-2 LNA nucleotides, andeach D is a sequence of 1-2 DNA nucleotides;

L-D-L-D-L-D-L, where each L is a sequence of 1-2 LNA nucleotides, andeach D is a sequence of 1-3 DNA nucleotides;

L-D-L-D-L-D-L-D-L, where each L is a sequence of 1-2 LNA nucleotides,and each D is a sequence of 1-3 DNA nucleotides; and

L-D-L-D-L-D-L-D-L, where each L is a sequence of 1-2 LNA nucleotides,and each D is a sequence of 1-2 DNA nucleotides.

The subject oligonucleotide sequences may further include one or moremodified internucleoside linkages, such as phosphorothioate,phosphorodithioate, phosphoramidate and/or thiophosphoramidate linkages.A non-naturally occurring internucleoside linkage may be included at anyconvenient position(s) of the sequence of the subject oligonucleotide.

In certain embodiments, the oligonucleotide has one of the followingsequences where capitalized letters denote LNA nucleotides and lowercaseletters denote DNA nucleotides (i.e., deoxyribonucleotide units):

(SEQ ID NO: 67) LNA 1: 5′ AccAaaAGaaT 3′; (SEQ ID NO: 68) LNA 2: 5′TggCcATcaaT 3′; (SEQ ID NO: 69) LNA 3: 5′ TagCAtActtA 3′;(SEQ ID NO: 70) LNA 4: 5′ CCAAAAGA 3′; (SEQ ID NO: 71) LNA 5: 5′CATACTTA 3′; (SEQ ID NO: 72) LNA 6: 5′ CagaCaCGaCCaaAA 3′;(SEQ ID NO: 73) LNA 7: 5′ TAcTtaCTgaCagCC 3′; (SEQ ID NO: 74) LNA 8: 5′AGACacgaccaAAAG 3′; (SEQ ID NO: 75) LNA 9: 5′ TACTtactgacaGCC 3′;(SEQ ID NO: 76) LNA9.2: 5′ TACttactgacAGCC 3′; (SEQ ID NO: 77) LNA10:5′ACCaaaagAAT 3′; (SEQ ID NO: 78) LNA11: 5′ TGGccatcAAT 3′;(SEQ ID NO: 79) LNA12: 5′TAGcatacTTA 3′; (SEQ ID NO: 80) LNA13:5′CgacCAaaAGaattC 3′; (SEQ ID NO: 81) LNA14: 5′CGACcaaaagaATTC 3′;(SEQ ID NO: 82) LNA15: 5′GaTGgCcATcaAttA 3′; (SEQ ID NO: 83) LNA16:5′GATGgccatcaATTA 3′; (SEQ ID NO: 84) LNA17: 5′TcTAgCaTActTacT 3′;(SEQ ID NO: 85) LNA18: 5′TCTAgcatactTACT 3′; (SEQ ID NO: 86) LNA19:5′GAAttcggatgGCCA 3′; (SEQ ID NO: 87) LNA20: 5′GGCCatcaattaGTG 3′;(SEQ ID NO: 88) LNA21: 5′TTCGgatggccaTCA 3′; (SEQ ID NO: 89) LNA22:5′AGCCagacagCGA 3′; and (SEQ ID NO: 90) LNA23: 5′GACAgccagacaGCA 3′.

Sequence mutants of the oligonucleotide sequences described above arealso encompassed by the present disclosure. It is understood that in anyof the sequences described herein that 1, 2, 3, 4 or more of thenucleotide may be mutated to provide for a desirable property, such asenhanced inhibition activity, conjugation to a modifying agent, etc.

In some cases, any one of the sequences described herein (e.g., one ofSEQ ID NO: 45-96) is comprised in a longer sequence, e.g., includesadditional 5′ and/or 3′ nucleotides. In certain instances, the subjectoligonucleotide is 30 nucleotides or less in length, such as 25 or less,20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less,14 or less, 13 or less, 12 or less, 11 or less, or 10 nucleotides orless in length.

In some cases, the subject oligonucleotide compound comprises a sequencehaving a deletion relative to one of the sequences described herein(e.g., one of SEQ ID NO: 45-96). For example, a sequence where 1, 2 or 3nucleotides are deleted from the 5′ and/or 3′ terminal of the sequence,e.g., one of SEQ ID NOs: 45-96. In some cases, the deletion sequence hasone nucleotide missing from the 5′ terminal of one of SEQ ID NOs: 45-96.In some cases, the deletion sequence has one nucleotide missing from the3′ terminal of one of SEQ ID NOs: 45-96. In some cases, the deletionsequence has two nucleotides missing from the 5′ terminal of one of SEQID NOs: 45-96. In some cases, the deletion sequence has two nucleotidesmissing from the 3′ terminal of one of SEQ ID NOs: 45-96.

In certain cases, the oligonucleotide sequence can include a mutationdesigned to cover single-nucleotide polymorphisms (SNPs) in a targetPSL2 sequence. In some cases, the oligonucleotide is a modified versionof LNA9 with single mutation sites that protect against PLS2 targetsequences containing a nucleotide change with the LNA9 target sequence.It is understood that SNP mutations of interest can be applied to any ofthe sequences described herein. Mutated sequences of interest include,but are not limited to the following:

(SEQ ID NO: 91) 5′ TACTTACTGACAGTC 3′; (SEQ ID NO: 92) 5′TACTTACCGACAGCC 3′; and (SEQ ID NO: 93) 5′ GGATTTCGGATGGCCA 3′.

In certain embodiments, the oligonucleotide has one of the followingsequences where capitalized letters denote LNA nucleotides and lowercaseletters denote DNA nucleotides:

(SEQ ID NO: 94) LNA9.G74C: 5′ TACTtactgacaGTC 3′; (SEQ ID NO: 95)LNA9.T80C: 5′ TACTtaccgacaGCC 3′; and (SEQ ID NO: 96) LNA19.U56C: 5′GGATttcggatggCCA 3′.

In certain instances, the oligonucleotide has a maximum length thatcorresponds to the particular region of the PSL2 structure (e.g., asub-region corresponding to nucleotides 34-87). In certain instances,the oligonucleotide length is 20 nucleotides or less, such as 15nucleotides or less, 14 nucleotides or less, 13 nucleotides or less, 12nucleotides or less, 11 nucleotides or less, 10 nucleotides or less, 9nucleotides or less, 8 nucleotides or less, 7 nucleotides or less, oreven less.

Oligonucleotides may be chemically synthesized by methods known in theart (see Wagner et al. (1993), supra, and Milligan et al., supra.)Oligonucleotides may be chemically modified from the nativephosphodiester structure, in order to increase their intracellularstability and binding affinity. A number of such modifications have beendescribed in the literature, which alter the chemistry of the backbone,sugars or heterocyclic bases.

Among useful changes in the backbone chemistry are phosphorothioates;phosphorodithioates, where both of the non-bridging oxygens aresubstituted with sulfur; phosphoroamidites; alkyl phosphotriesters andboranophosphates. Achiral phosphate derivatives include3′-O′-5′-S-phosphorothioate, 3′-S-5′-O-phosphorothioate,3′-CH₂-5′-O-phosphonate, 3′-NH-5′-O-phosphoroamidate, andthiophosphoramidates. Peptide nucleic acids replace the entire ribosephosphodiester backbone with a peptide linkage. Sugar modifications arealso used to enhance stability and affinity. The α-anomer of deoxyribosemay be used, where the base is inverted with respect to the naturalβ-anomer. The 2′-OH of the ribose sugar may be altered to form2′-O-methyl or 2′-O-allyl sugars, which provides resistance todegradation without comprising affinity. Modification of theheterocyclic bases must maintain proper base pairing. Some usefulsubstitutions include deoxyuridine for deoxythymidine;5-methyl-2′-deoxycytidine and 5-bromo-2′-deoxycytidine fordeoxycytidine. 5-propynyl-2′-deoxyuridine and5-propynyl-2′-deoxycytidine have been shown to increase affinity andbiological activity when substituted for deoxythymidine anddeoxycytidine, respectively.

The oligonucleotide agents may be derivatized with any convenientmodifying agent, e.g., by conjugation of the modifying agent to the 5′-and/or 3′terminal of the oligonucleotide sequence. In some cases, themodifying agent is a moiety that enhances cellular uptake (e.g., alipid). Any convenient lipids may be conjugated to the subjectoligonucleotides. In some instances, the modifying agent is a fattyacid, connected to the 5′ or 3′ terminal via an optional linker. Thelipid group can be an aliphatic hydrocarbon or fatty acid, including butnot limited to, derivatives of hydrocarbons and fatty acids, withexamples being saturated straight chain compounds having 14-20 carbons,such as myristic (tetradecanoic) acid, palmitic (hexadecanoic) acid, andstearic (octadeacanoic) acid, and their corresponding aliphatichydrocarbon forms, tetradecane, hexadecane and octadecane. Examples ofother suitable lipid groups that may be employed are sterols, such ascholesterol, and substituted fatty acids and hydrocarbons, particularlypolyfluorinated forms of these groups. The scope of the lipid groupincludes derivatives such as amine, amide, ester and carbamatederivatives.

In some cases, the modifying agent is a further nucleic acid sequencehaving a desirable activity (e.g., recruitment of an RNase, as describedherein). In certain instances, the modifying agent has a specificbinding activity that provides for delivery of the oligonucleotide to aparticular target, such as a cell-specific protein. In some cases, themodifying agent is an antibody of interest that specifically binds acell-specific target of interest. In certain instances, the antibodymodifying agent is specifically binds a hemagglutinin (HA) target.

The oligonucleotide active agent can be utilized in any convenient form.In some instances, the oligonucleotide active agent is single stranded.In some instances, the oligonucleotide active agent is double stranded.In some instances, the oligonucleotide active agent is an siRNA. In someinstances, the oligonucleotide active agent is an shRNA. In someinstances, the oligonucleotide active agent is a ssRNA. In someinstances, one or more nucleotides of the ssRNA can be replaced with LNAnucleotides. In some instances, the oligonucleotide active agent is assDNA. In some instances, one or more nucleotides of the ssDNA can bereplaced with LNA nucleotides.

Methods of Treatment

Aspects of the present disclosure include methods of treating orpreventing influenza A virus infection in a subject. The subjectoligonucleotide compounds find use as a new class of antiviraltherapeutics that can efficiently disrupt packaging and completelyprevent otherwise lethal disease in vivo. As demonstrated in theexamples section, in vivo, intranasal dosing of exemplaryoligonucleotide compounds resulted in potent antiviral efficacy andprevented lethal IAV infection in mice.

Aspects of the method include administering to a subject in need thereofa therapeutically effective amount of a subject compound to treat thesubject for an infection or prevent infection in the subject. By “atherapeutically effective amount” is meant the concentration of acompound that is sufficient to elicit the desired biological effect(e.g., treatment or prevent of the condition or disease, influenza Avirus infection). By “treatment” is meant that at least an ameliorationof the symptoms associated with the condition afflicting the host isachieved, where amelioration is used in a broad sense to refer to atleast a reduction in the magnitude of a parameter, e.g. symptom,associated with the condition being treated. As such, treatment alsoincludes situations where the pathological condition, or at leastsymptoms associated therewith, are completely inhibited, e.g., preventedfrom happening, or stopped, e.g. terminated, such that the host nolonger suffers from the condition, or at least the symptoms thatcharacterize the condition. Thus treatment includes: (i) prevention,that is, reducing the risk of development of clinical symptoms,including causing the clinical symptoms not to develop, e.g., preventingdisease progression to a harmful state; (ii) inhibition, that is,arresting the development or further development of clinical symptoms,e.g., mitigating or completely inhibiting an active disease (e.g.,infection); and/or (iii) relief, that is, causing the regression ofclinical symptoms. In the context of influenza A virus infection, theterm “treating” includes any or all of: reducing the number of viralcells in patient samples, inhibiting replication of viral cells, andameliorating one or more symptoms associated with an infection.

The subject to be treated can be one that is in need of therapy, wherethe host to be treated is one amenable to treatment using the subjectcompound. In some embodiments, the subject is one that is suspected ofhaving an influenza A virus infection. In certain embodiments, thesubject is diagnosed as having an influenza A virus infection. As such,in some cases, the subject is one who has been infected with the virus.

In certain cases, the subject is one who is at risk of being infected,or is suspected of being infected with the virus. In some embodiments,the vRNA is a PB2 vRNA. The subject methods can be used to preventinfection of the subject with an influenza A virus. By “prevention” ismeant that the subject at risk of influenza A virus infection is notinfected despite exposure to the virus under conditions that wouldnormally lead to infection. In some cases, the administering of thesubject active agent (e.g., oligonucleotide compound) protects thesubject against infection for 1 week or more, such as2 weeks or more, 3weeks or more, 1 month or more, 2 months or more, 3 months or more, etc.Multiple doses of the subject compound can be administered according tothe subject methods to provide for protection of the subject forminfection for an extended period of time. The timing and dosage amountscan be readily determined using conventional methods.

In some cases, the subject methods of treatment include a step ofdetermining or diagnosing whether the subject has a influenza A virusinfection. The determining step can be performed using any convenientmethods. In some cases, the determining step includes obtaining abiological sample from the subject and assaying the sample for thepresence of viral cells. The sample can be a cellular sample. Thedetermining step can include identification of viral cells including aparticular mutation.

Accordingly, a variety of subjects may be amenable to treatment usingthe subject compounds and pharmaceutical compositions disclosed herein.As used herein, the terms “subject” and “host” are used interchangeably.Generally, such subjects are “mammals”, with humans being of interest.Other subjects can include domestic pets (e.g., dogs and cats),livestock (e.g., cows, pigs, goats, horses, and the like), rodents(e.g., mice, guinea pigs, and rats, e.g., as in animal models ofdisease), as well as non-human primates (e.g., chimpanzees, andmonkeys).

The amount of the subject compound administered can be determined usingany convenient methods to be an amount sufficient to produce the desiredeffect in association with a pharmaceutically acceptable diluent,carrier or vehicle. The specifications for the unit dosage forms of thepresent disclosure will depend on the particular compound employed andthe effect to be achieved, and the pharmacodynamics associated with eachcompound in the host.

In some embodiments, an effective amount of subject compound is anamount that ranges from about 50 ng/ml to about 50 μg/ml (e.g., fromabout 50 ng/ml to about 40 μg/ml, from about 30 ng/ml to about 20 μg/ml,from about 50 ng/ml to about 10 μg/ml, from about 50 ng/ml to about 1μg/ml, from about 50 ng/ml to about 800 ng/ml, from about 50 ng/ml toabout 700 ng/ml, from about 50 ng/ml to about 600 ng/ml, from about 50ng/ml to about 500 ng/ml, from about 50 ng/ml to about 400 ng/ml, fromabout 60 ng/ml to about 400 ng/ml, from about 70 ng/ml to about 300ng/ml, from about 60 ng/ml to about 100 ng/ml, from about 65 ng/ml toabout 85 ng/ml, from about 70 ng/ml to about 90 ng/ml, from about 200ng/ml to about 900 ng/ml, from about 200 ng/ml to about 800 ng/ml, fromabout 200 ng/ml to about 700 ng/ml, from about 200 ng/ml to about 600ng/ml, from about 200 ng/ml to about 500 ng/ml, from about 200 ng/ml toabout 400 ng/ml, or from about 200 ng/ml to about 300 ng/ml).

In some embodiments, an effective amount of a subject compound is anamount that ranges from about 10 μg to about 100 mg, e.g., from about 10pg to about 50 pg, from about 50 pg to about 150 pg, from about 150 pgto about 250 pg, from about 250 pg to about 500 pg, from about 500 pg toabout 750 pg, from about 750 pg to about 1 ng, from about 1 ng to about10 ng, from about 10 ng to about 50 ng, from about 50 ng to about 150ng, from about 150 ng to about 250 ng, from about 250 ng to about 500ng, from about 500 ng to about 750 ng, from about 750 ng to about 1 μg,from about 1 μg to about 10 μg, from about 10 μg to about 50 μg, fromabout 50 μg to about 150 μg, from about 150 μg to about 250 μg, fromabout 250 μg to about 500 μg, from about 500 μg to about 750 μg, fromabout 750 μg to about 1 mg, from about 1 mg to about 50 mg, from about 1mg to about 100 mg, or from about 50 mg to about 100 mg. The amount canbe a single dose amount or can be a total daily amount. The total dailyamount can range from 10 pg to 100 mg, or can range from 100 mg to about500 mg, or can range from 500 mg to about 1000 mg.

In some embodiments, a single dose of the subject compound isadministered. In other embodiments, multiple doses of the subjectcompound are administered. Where multiple doses are administered over aperiod of time, the subject compound can be administered twice daily(qid), daily (qd), every other day (qod), every third day, three timesper week (tiw), or twice per week (biw) over a period of time. Forexample, a compound can be administered qid, qd, qod, tiw, or biw over aperiod of from one day to about 2 years or more. For example, a compoundcan be administered at any of the aforementioned frequencies for oneweek, two weeks, one month, two months, six months, one year, or twoyears, or more, depending on various factors.

Any of a variety of methods can be used to determine whether a treatmentmethod is effective. For example, a biological sample obtained from anindividual who has been treated with a subject method can be assayed forthe presence and/or level of viral cells. Assessment of theeffectiveness of the methods of treatment on the subject can includeassessment of the subject before, during and/or after treatment, usingany convenient methods. Aspects of the subject methods further include astep of assessing the therapeutic response of the subject to thetreatment.

In some embodiments, the method includes assessing the condition of thesubject, including diagnosing or assessing one or more symptoms of thesubject which are associated with the disease or condition of interestbeing treated (e.g., as described herein). In some embodiments, themethod includes obtaining a biological sample from the subject andassaying the sample, e.g., for the presence of viral cells or componentsthereof that are associated with the disease or condition of interest(e.g., as described herein). The sample can be a cellular sample. Theassessment step(s) of the subject method can be performed at one or moretimes before, during and/or after administration of the subjectcompounds, using any convenient methods. In certain cases, theassessment step includes identification and/or quantitation of viralcells. In certain instances, assessing the subject include diagnosingwhether the subject has a viral infection or symptoms thereof.

Screening Methods

Aspects of the present disclosure also include screening assaysconfigured to identify agents that find use in methods of the invention,e.g., as reviewed above. Aspects of the present disclosure includemethods for screening a candidate agent for the ability to inhibitinfluenza A virus in a cell. In some instances, the method comprises:contacting a sample comprising viral RNA (vRNA) comprising a PSL2 motifwith a candidate agent; and determining whether the candidate agentspecifically binds to the PSL2 motif. In some cases, an agent thatspecifically binds to the PSL2 motif will treat the subject having theinfluenza A virus infection. By assessing or determining is meant atleast predicting that a given test compound will have a desirableactivity, such that further testing of the compound in additionalassays, such as animal model and/or clinical assays, is desired.

The candidate agent is selected from: a small molecule, anoligonucleotide, an antibody and a polypeptide. In some instances, thedetermining step comprises detecting a cellular parameter, wherein achange in the parameter in the cell as compared to in a cell notcontacted with candidate agent indicates that the candidate agentspecifically binds the PSL2 motif. In some cases, the subject screeningmethod is a method of SHAPE analysis (Selective 2′-hydroxyl acylationanalyzed by primer extension). In certain instances, the candidate agentis an oligonucleotide.

Drug screening may be performed using an in vitro model, a geneticallyaltered cell or animal, or purified PSL2 protein. One can identifyligands that compete with, modulate or mimic the action of a lead agent.Drug screening identifies agents that bind to particular sites of PSL2motif. A wide variety of assays may be used for this purpose, includinglabeled in vitro binding assays, electrophoretic mobility shift assays,immunoassays for protein binding, and the like. Knowledge of the3-dimensional structure of PSL2, derived from the structural studiesdescribed herein, can also lead to the rational design of small drugsthat specifically inhibit IAV activity.

The term “agent” as used herein describes any molecule, e.g.,oligonucleotide, protein or pharmaceutical, with the capability ofbinding PSL2 to inhibit IAV. Generally, a plurality of assay mixturesare run in parallel with different agent concentrations to obtain adifferential response to the various concentrations. Typically one ofthese concentrations serves as a negative control, i.e., at zeroconcentration or below the level of detection.

Candidate agents encompass numerous chemical classes, such asoligonucleotides, antibodies, polypeptides, and organic molecules, e.g.,small organic compounds having a molecular weight of more than 50 andless than about 2,500 daltons. Candidate agents comprise functionalgroups necessary for structural interaction with proteins, particularlyhydrogen bonding, and typically include at least an amine, carbonyl,hydroxyl or carboxyl group, preferably at least two of the functionalchemical groups. The candidate agents often comprise cyclical carbon orheterocyclic structures and/or aromatic or polyaromatic structuressubstituted with one or more of the above functional groups. Candidateagents are also found among biomolecules including peptides,saccharides, fatty acids, steroids, purines, pyrimidines, derivatives,structural analogs or combinations thereof.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides and oligopeptides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs. Of interest in certain embodiments are compoundsthat pass the blood-brain barrier.

Where the screening assay is a binding assay, one or more of themolecules may be joined to a member of a signal producing system, e.g.,a label, where the label can directly or indirectly provide a detectablesignal. Various labels include, but are not limited to: radioisotopes,fluorescers, chemiluminescers, enzymes, specific binding molecules,particles, e.g., magnetic particles, and the like. Specific bindingmolecules include pairs, such as biotin and streptavidin, digoxin andantidigoxin, etc. For the specific binding members, the complementarymember would normally be labeled with a molecule that provides fordetection, in accordance with known procedures.

A variety of other reagents may be included in the screening assay.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc. that are used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Reagentsthat improve the efficiency of the assay, such as protease inhibitors,nuclease inhibitors, anti-microbial agents, etc. may be used. Themixture of components is added in any order that provides for therequisite binding. Incubations are performed at any suitabletemperature, typically between 4 and 40° C. Incubation periods areselected for optimum activity, but may also be optimized to facilitaterapid high-throughput screening. Typically between 0.1 and 1 hours willbe sufficient.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Materials and Methods

Cells and Viruses:

HEK 293T and MDCK cells were obtained from American Type CultureCollection (Manasass, Va.) and were maintained in Dulbecco's modifiedEagle's medium with 10% fetal bovine serum and penicillin-streptomycin(Gibco). Influenza A/PR/8/34 (PR8) H1N1 virus was generated using aneight-plasmid reverse genetic system. Tissue-cultured adapted influenzaA/Hong Kong/8/68 (HK68) H3N2 virus was obtained from ATCC(ATCC-VR-1679). Viruses were grown and amplified in 10-day-oldspecific-pathogen-free chicken embryos at 35° C. (Charles RiverLaboratories; SPAEAS).

Plasmid Constructs and Cloning:

Plasmids were used containing the wild-type PB2 segments from influenzaviruses A/PuertoRico/8/34 (H1N1) [PR8], A/New York/470/2004 (H3N2)[NY470], A/New York/312/2001 (H1N1) [NY312], A/Brevig Mission/1/1918(H1N1) [1918], A/California/04/2009 (H1N1) [CA09], A/Vietnam/03/2004(H5N1) [VN1203], and A/Anhui/1/2013 (H7N9). For the generation of PR8packaging mutant vRNA, we utilized a Stratagene QuickChange XLsite-directed mutagenesis kit (Stratagene) for mutagenesis of a pDZplasmid containing the PB2 gene of PR8. Sequences of each mutatedconstruct were confirmed by automated sequencing.

Reverse Genetics and Virus Titrations:

Influenza A/Puerto Rico/8/34 (PR8) virus was generated using aneight-plasmid reverse genetic system (Hoffman et al., 2000). Briefly, toproduce recombinant PR8 viruses, 10⁶ cells of a 293T/MDCK co-culturewere Lipofectamine 3000 (Invitrogen) transfected with 1 ug of one ofeach of the eight segments contained within plasmids that utilize abidirectional dual Pol I/II promoter system for the simultaneoussynthesis of genomic vRNA and mRNA. Cells were collected 24 hourspost-transfection and inoculated into the allantoic cavities of10-day-old chicken embryos (Charles River, research gradespecific-pathogen-free eggs). Rescue of recombinant viruses was assessedby hemagglutination activity. Each newly rescued virus was furtherplaque titered and mutations were confirmed by sequencing of mutatedgenes. Plaque assays were carried out on confluent MDCK cells asdescribed previously (Szretter et al., 2006). Hemagglutination (HA)assays were carried out in 96-well round-bottomed plates atroom-temperature, using 50 ul of virus dilution and 50 ul of a 0.5%suspensions of turkey red blood cells in phosphate-buffered saline(PBS).

Viral Growth Kinetics:

Growth kinetics for PR8 viruses was determined by inoculation of10-day-old chicken eggs with 100 plaque-forming units (PFU) of virus. At72 h post-inoculation, the virus titer in the allantoic fluid wasdetermined by titration of plaques on MDCK cells.

Isolation of Packaged vRNAs:

To analyze packaged vRNA for PR8 mutated viruses, 10-day-old eggs wereinoculated with approximately 1000 PFU of recombinant virus andincubated for 72 h. Allantoic fluid was harvested, and supernatant wasclarified by low-speed centrifugation. Clarified supernatant was thenlayered on a 30% sucrose cushion and ultra-centrifuged at 30,000 RPM for2.5 h (Beckman Rotor SW41). Pelleted virus was resuspended in PBS andTRIzol (Invitrogen) extracted. Precipitated vRNA was resuspended in afinal volume of 20 ul of 10 mM Tris-HCl (pH 8.0) and stored at −80° C.

qPCR Analysis of Packaged vRNAs:

Approximately 200 ng of extracted vRNAs were reverse transcribed using auniversal 3′ primer (5′-AGGGCTCTTCGGCCAGCRAAAGCAGG) (SEQ ID NO:97) andSuperscript III reverse transcriptase (RT) (Invitrogen). The RT productwas diluted 10,000-fold and used as a template for quantitative PCR(qPCR). Separate PCRs were then carried out as previously described(Marsh et al., 2007) with segment-specific primers. The 10 ul reactionmixture contained 1 ul of diluted RT product, a 0.5 uM primerconcentration, and SYBR Select Master Mix (Applied Biosystems) thatincluded SYBR GreenER dye, 200 uM deoxynucleoside triphosphates,heat-labile UDG, optimized SYBR Green Select Buffer, and AmpliTaq DNApolymerase UP enzyme. Relative vRNA concentrations were determined byanalysis of cycle threshold values, total vRNA amount was normalized byequalization of the level of HA vRNA, and then percentages ofincorporation were calculated relative to the levels of wt vRNApackaging. Viral packaging results represent the averaged levels of vRNAincorporation±standard deviations derived from two independent viruspurifications, with vRNA levels quantified in triplicate, n=6.

Mouse Infections:

Groups of 6-8 week old female BALB/C mice (Jackson Laboratory) werelightly anesthetized with isoflurane and intranasally infected with 50ul of 1000 PFU of wild type mouse-adapted PR8 (H1N1) virus (ATCC), PB2mutated PR8 recombinant viruses, or sterile PBS. Weights were measureddaily, and animals were humanely sacrificed by day 10 or when weightloss exceeded 20%. All animal care and experimental procedures are inaccordance with the National Institutes of Health Guidelines for theCare and Use of Laboratory Animals and approved by the StanfordUniversity Administrative Panel on Laboratory Animal Care.

Locked Nucleic Acid (LNA) Design and Preparation:

Oligonucleotides containing locked nucleic acids (LNA) were customsynthesized from Exiqon. Capitalized letters denote LNA. Lowercaseletters denote typical (non-locked) DNA nucleotides. Alloligonucleotides contained phosphorothioate internucleoside linkages.The LNAs were designed to be complementary to different sequencescontained in the PSL2 structure of segment PB2. LNA 8 and 9 are designedto contain a stretch of 6-8 DNA nucleotides for RNAse-H recruitment.Sequences of all LNAs are shown below.

(SEQ ID NO: 67) LNA 1: 5′ AccAaaAGaaT 3′ (SEQ ID NO: 68) LNA 2: 5′TggCcATcaaT 3′ (SEQ ID NO: 69) LNA 3: 5′ TagCAtActtA 3′ (SEQ ID NO: 70)LNA 4: 5′ CCAAAAGA 3′ (SEQ ID NO: 71) LNA 5: 5′ CATACTTA 3′(SEQ ID NO: 72) LNA 6: 5′ CagaCaCGaCCaaAA 3′ (SEQ ID NO: 73) LNA 7: 5′TAcTtaCTgaCagCC 3′ (SEQ ID NO: 74) LNA 8: 5′ AGACacgaccaAAAG 3′-with RNase-H activity (SEQ ID NO: 75) LNA 9: 5′ TACTtactgacaGCC 3′-with RNase-H activity (SEQ ID NO: 76) LNA9.2: 5′ TACttactgacAGCC 3′(SEQ ID NO: 77) LNA10: 5′ACCaaaagAAT 3′ (SEQ ID NO: 78) LNA11: 5′TGGccatcAAT 3′ (SEQ ID NO: 79) LNA12: 5′TAGcatacTTA 3′ (SEQ ID NO: 80)LNA13: 5′CgacCAaaAGaattC 3′ (SEQ ID NO: 81) LNA14: 5′CGACcaaaagaATTC 3′(SEQ ID NO: 82) LNA15: 5′GaTGgCcATcaAttA 3′ (SEQ ID NO: 83) LNA16:5′GATGgccatcaATTA 3′ (SEQ ID NO: 84) LNA17: 5′TcTAgCaTActTacT 3′(SEQ ID NO: 85) LNA18: 5′TCTAgcatactTACT 3′ (SEQ ID NO: 86) LNA19:5′GAAttcggatgGCCA 3′ (SEQ ID NO: 87) LNA20: 5′GGCCatcaattaGTG 3′(SEQ ID NO: 88) LNA21: 5′TTCGgatggccaTCA 3′ (SEQ ID NO: 89) LNA22:5′AGCCagacagCGA 3′ (SEQ ID NO: 90) LNA23: 5′GACAgccagacaGCA 3′

The following oligonucleotides were designed to cover single-nucleotidepolymorphisms (SNPs) in PSL2 sequence. The following exemplary sequencesare modified versions of LNA9 with single mutation sites that wouldprotect against a few avian and bat strains whose PLS2 sequence containsa nucleotide change with the LNA9 target sequence. It is understood thatsimilar design can be applied to any of the sequences described herein.

(SEQ ID NO: 94) LNA9.G74C: 5′ TACTtactgacaGTC 3′ (SEQ ID NO: 95)LNA9.T80C: 5′ TACTtaccgacaGCC 3′ (SEQ ID NO: 96) LNA19.U56C: 5′GGATttcggatggCCA 3′

Antiviral Assays:

LNAs were reconstituted in RNAse free water at 100 μM, aliquoted andstored at −20° C. prior to single use. Lipofectamine 3000 (LifeTechnology) was used to transfect LNA into cells at a finalconcentrations of 1 μM, 100 nM, 10 nM, and 1 nM per manufacturer'sprotocol. For prophylactic antiviral assays, 10⁶ MDCK cells were platedin 6-well plates 24 h prior to being transfected with the indicated LNA.Cells were then infected at 4 h, 2 h, or 1 h post-transfection with 0.01MOI of PR8 (H1N1) or HK68 (H3N2) virus. For post-infection, therapeuticantiviral assessment, MDCK cells were infected with PR8 or HK68 asdescribed. LNAs were then transfected at 4 h, 2 h, or 1 hpost-infection. After 48 h post-infection, supernatant was collected,and viral titer was determined by plaque assay.

In Vitro Transcription of vRNA:

For each wild-type isolate (PR8, 1918, VN1203, NY470, NY312, CA09, andA/Anhui/1/2013 H7N9) and PR8 packaging mutant clones, PB2 cDNA wasamplified from plasmid using segment-specific primers under a T7promoter. Amplified cDNA was gel-purified using an Invitrogen DNA gelkit. vRNAs were then produced by in vitro transcription, usingT7-MEGAscript. vRNAs for SHAPE were purified by MEGAclear (Thermofisher,cat. no. AM1908) with purity and length verified by capillaryelectrophoresis.

Sf-SHAPE Analysis of vRNA:

PB2 vRNA was folded (100 mM NaCl; 2.5 mM MgCl; 65° C. for 1 min, 5 mincooling at room temperature, 37° C. for 20-30 min) in 100 mM HEPES,pH=8. 2 min acylation with NMIA (Wilkinson et al., 2006) and reversetranscription (RT) primer extension were performed at 45° C. for 1 min,52° C. for 25 min, 65° C. for 5 min, as previously described (Mortimerand Weeks, 2009). 6FAM was used for all labeled primers. Exceptions tothese protocols were as follows: (i) RNA purification after acylationwas performed using RNA C&C columns (Zymo Research), rather than ethanolprecipitation; (ii) before and after SHAPE primer buffer was added, themixture was placed at room temperature for 2-5 min, which enhanced RTtranscription yields significantly; (iii) DNA purification was performedusing Sephadex G-50 size exclusion resin in 96-well format thenconcentrated by vacuum centrifugation, resulting in a more significantremoval of primer; and (iv) 2 pmol RNA was used in ddGTP RNA sequencingreactions.

The ABI 3100 Genetic Analyzer (50 cm capillaries filled with POP6matrix) was set to the following parameters: voltage 15 kV, T=60° C.,injection time=15 s. The GeneScan program was used to acquire the datafor each sample, which consisted of purified DNA resuspended in 9.75 ulof Hi-Di formamide, to which 0.25 ul of ROX 500 internal size standard(ABI Cat. 602912) was added. PeakScanner parameters were set to thefollowing parameters: smoothing=none; window size=25; size calling=localsouthern; baseline window=51; peak threshold=15. Fragments 250 and 340were computationally excluded from the ROX500 standard (Akbari et al.,2008). The data from PeakScanner were then processed into SHAPE data byusing FAST (fast analysis of SHAPE traces), a custom program (Pang etal., 2011). FAST automatically corrects for signal differences due tohandling errors, adjusts for signal decay, and converts fragment lengthto nucleotide position, using a ddGTP ladder as an external sizingstandard and the local Southern method (Pang et al., 2011 and Pang etal., 2012).

RNAstructure parameters: slope and intercept parameters of 2.6 and −0.8kcal/mol, were initially tried, as suggested (Deigan et al., 2009);smaller intercepts closer to 0.0 kcal/mol (e.g. ˜−0.3) were found toproduce fewer less optimal structures (within a maximum energydifference of 10%). This minor parameter difference may be due to theprecise fitting achieved between experimental and control data sets bythe automated FAST algorithm. In its current implementation, FAST isintegrated into RNAstructure, which requires MFC (Microsoft FoundationClasses). RNA structures were drawn and colored using RNAViz 2 (De Rijket al., 2003) and finalized in Adobe Illustrator.

Construct Design, RNA Synthesis and Chemical Modification forMutate-and-Map Experiments:

Double-stranded DNA templates were prepared by PCR assembly of DNAoligomers designed by an automated MATLAB script as previously described(NA_Thermo, available at “https:” followed by “//github.” Followed by“com/DasLab/NA_thermo”) (Kladwang and Cordero et al., 2011). Constructsfor mutate-and-map (M²) includes all single mutants to Watson-Crickcounterpart. Compensatory mutants for mutation/rescue were designedbased on base-pairing in the proposed secondary structure (Tian et al.,2014). In vitro transcription reactions, RNA purification andquantification steps were as described previously (Kladwang and Corderoet al., 2011). One-dimensional chemical mapping, mutate-and-map (M²),and mutation/rescue were carried out in 96-well format as describedpreviously (Kladwang and VanLang et al., 2011; Kladwang and Cordero etal., 2011; Cordero et al., 2013). Briefly, RNA was heated up and cooledto remove secondary structure heterogeneity; then folded properly andincubated with SHAPE reagent (5 mg/mL 1-methyl-7-nitroisatoic anhydride(1M7)) (Mortimer and Weeks, 2007); modification reaction was quenchedand RNA are recovered by poly(dT) magnetic beads (Ambion) andFAM-labeled Tail2-A20 primer; RNA was washed by 70% ethanol (EtOH) twiceand resuspended in ddH₂O; followed by reverse transcription to cDNA andheated NaOH treatment to remove RNA. Final cDNA library was recovered bymagnetic bead separation, rinsed, eluted in Hi-Di formamide (AppliedBiosystems) with ROX-350 ladder, loaded to capillary electrophoresissequencer (ABI3100). Data processing, structural modeling, and datadeposition: The HiTRACE software package version 2.0 was used to analyzeCE data (both MATLAB toolbox and web server available (Yoon et al.,2011; Kim et al., 2013)). Trace alignment, baseline subtraction,sequence assignment, profile fitting, attenuation correction andnormalization were accomplished as previously described (Kim et al.,2009; Kladwang et al., 2014). Sequence assignment was accomplishedmanually with verification from sequencing ladders. Data-drivensecondary structure models were obtained using the Fold program of theRNAstructure package version 5.4 (Mathews et al., 2004) withpseudo-energy slope and intercept parameters of 2.6 kcal/mol and −0.8kcal/mol. 2-dimensional Z score matrices for M² datasets, and helix-wisebootstrapping confidence values were calculated as described previously(Tian et al., 2014; Kladwang and VanLang et al., 2011). Z score matriceswere used as base-pair-wise pseudofree energies with a slope andintercept of 1.0 kcal/mol and 0 kcal/mol. Secondary structure imageswere generated by VARNA (Darty et al., 2009). All chemical mappingdatasets, including one-dimensional mapping, mutate-and-map, andmutation/rescue, have been deposited at the RNA Mapping Database(“http:” followed by “//rmdb.stanford.” followed by “edu”) (Cordero etal., 2012).

SHAPE Analysis of LNA-Targeted vRNA:

DNA template of PR8 segment PB2 was prepared by PCR assembly of DNAoligomers, and in vitro transcription reactions, RNA purification andquantification steps were as described previously (Kladwang and VanLanget al., 2011). One-dimensional SHAPE chemical mapping was performed in96-well plate format as described above with the following exception:once RNA was denatured and refolded as described, 100 nM of eachprepared LNA was added to the folded RNA and incubated with 5 mg/mL ofSHAPE reagent 1M7 (1-methyl-7-nitroisatoic anhydride). Modificationquenching, RNA recovery, re-suspension, reverse transcription, cDNAsequencing and data processing was performed as described, see, Kladwangand VanLang et al., 2011.

Example 1 SHAPE-Characterization of IAV Segment PB2 Packaging SignalIdentifies Conserved Structure

Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) andcomputational modeling was applied to IAV segment PB2 genomic vRNA tosearch for structured RNA domains. In vitro transcribed full-length(−)-sense PB2 vRNA from strain A/Puerto Rico/8/1934 (H1N1) “PR8” wasfolded in solution (Pang et al., 2011) and interrogated using anelectrophilic SHAPE reagent that preferentially reacts with nucleotidesexisting in flexible, single-stranded states (Wilkinson et al., 2006)(FIG. 1). This analysis revealed that much of the 2341-nt vRNA islargely unstructured (FIG. 2), consistent with recent bioinformaticsstudies that found higher potential for RNA secondary structureconservation in the (+)-sense over the (−)-sense RNA for all segments,including PB2 (Priore et al., 2012; Moss et al., 2011). These previousstudies did not analyze the terminal coding regions (TCR), and insteadstopped 80 nucleotides short of the PB2 5′ TCR's end. SHAPE-guidedmodeling suggested several areas in this region which contain stable RNAsecondary structures, most notably a stem-loop motif, named herein asthe Packaging Stem-Loop 2 (PSL2) (FIG. 1A), comprising nucleotides 34-87((−)-sense notation). This segment included a set of nucleotides thatwere previously implicated in PB2 packaging through mutational analysisvia an unidentified mechanism (FIG. 1A-1B, see circled nucleotides) (Gaoet al., 2012; Marsh et al., 2008; Liang et al., 2008; Gog et al., 2007).Supporting the hypothesis that these prior mutations act throughdisruption of PSL2 structure, SHAPE analysis of the mutants yieldeddifferent conformations that all abrogated the wild-type PSL2 structure(FIG. 1C, FIG. 3). The 60-nucleotide region encompassing PSL2 displaysnear 100% sequence conservation at the single nucleotide level betweenseasonal as well as pandemic strains of different subtypes and speciesorigins (FIG. 4), suggesting the existence of a strict biologicrequirement to maintain an intact PSL2 structure. Because differingdownstream sequences within PB2 vRNA could alter the secondary structureof PSL2, PSL2's structural conservancy was explored by performing SHAPEanalysis on full-length wild-type PB2 vRNAs isolated from a variety ofIAV strains and subtypes, including the highly pathogenic avian H5N1 andpandemic 1918 H1N1 strains. Despite the presence of two divergingnucleotides within the stem-loop and significant divergence in flankingsequences, the PSL2 stem-loop structure was recovered in SHAPE-guidedmodeling of PB2 RNA across these diverse species and subtypes (FIG.1D-1F).

FIG. 1A-FIG. 1F shows SHAPE-chemical mapping performed on full-length(−)-sense wild-type PB2 vRNAs. Colors denote SHAPE reactivity, which isproportional to the probability that a nucleotide is single-stranded.All structures are truncated to highlight the 5′ termini sequencestructure. Energy=AG free energy value of determined structuresgenerated by the RNAstructure modeling algorithm using SHAPE pseudofreeenergy parameters. (FIG. 1A) Wild-type PB2 RNA secondary structure fromstrain A/Puerto Rico/8/1934 “PR8” (H1N1). Color-coded circles correspondto nucleotides sites where synonymous mutations were reported to affectPB2 packaging (Gao et al., 2012; Marsh et al., 2011). (FIG. 1B)Packaging efficiency of synonymous mutants in (FIG. 1a ), determined byqPCR. Results performed in triplicate. Error bars=±SD. Box belowindicates mutant name and corresponding mutational change. Nucleotidenumbering shown in the genomic (−)-sense orientation. (FIG. 1C)SHAPE-determined structures of PB2 packaging-defective mutant vRNAs,m757 (G44C) and m745 (A80U). Black boxes=site(s) of synonymous mutation,(FIG. 1D-FIG. 1F) SHAPE-determined structures of wild-type PB2 frompandemic and highly pathogenic strains, including different subtypes:(FIG. 1D) 1918 pandemic (A/Brevig Mission/1/1918 (H1N1)), (FIG. 1E)Highly-pathogenic avian (A/Vietnam/1203/2004 (H5N1)), (FIG. 1F) 2009pandemic ‘swine’ (A/California/04/2009 (H1N1)).

FIG. 2, panels A-B, shows SHAPE reactivity of full-length PB2 vRNA.(FIG. 2, panel A) Average SHAPE reactivity (window bin size=100 nt) as afunction of nucleotide position for the full-length (−)-sense PB2 vRNAfrom IAV strain A/Puerto Rico/8/1934 (H1N1). The PSL2 region(highlighted in blue, nts 34-86) encompassess the 5′ packaging signaldomain, which possesses a high density of codons whose third position isconserved, and has one of the lowest SHAPE reactivities within the vRNA.The region after PSL2 is relatively unstructured yet contains anotherpotential site for the presence of RNA structure between nucleotides1400 and 1500. Interestingly, this second internal region was alsopredicted to contain structural elements by a 2011 bioinformatics study(ref. 19). (FIG. 2, panel B) Zoomed in view of PSL2 region from (FIG. 2a). Window bin size=10 nt.

FIG. 3A-FIG. 3E shows packaging-defective mutations disrupt wild-typeSHAPE reactivity. Left: Mutant SHAPE reactivity plotted as change overWT. Nucleotide numbering starts from 5′ end of (−)-sense vRNA. Orangebars indicate site of mutation. Energy values represent ΔG free energyof the predicted structure generated by the RNAstructure modelingalgorithm using SHAPE pseudofree energy parameters. Right:SHAPE-determined structures of full-length (−)-sense mutant PB2 vRNAfrom PR8 strain (H1N1). Images are truncated to highlight 5′ terminalregion. (FIG. 3A) Wild-type. Packaging-defective mutants: (FIG. 3B)m744b (AG83, 85UA). (FIG. 3C) m745 (A80U). (FIG. 3D) m55c (CU35, 36UC).(FIG. 3E) m757 (G44C).

FIG. 4 shows conservancy of nucleotide sequence containing the PSL2structure. Graphical representation of nucleic acid sequence alignmentsacross diverse influenza A viral subtypes and strains (“weblogo.”followed by “berkeley” followed by “.edu”). The overall heightrepresents sequence conservation at that nucleotide position, while theheight of symbols within each position indicates the relative frequencyof each nucleotide at that site. Black box=PSL2 region. Sequencesincluded in the alignment: highly pathogenic A/Brevig Mission/1/1918(H1N1), pandemic “swine flu” A/California/04/2009 (H1N1), modern humanA/New York/470/2004 (H3N2), human A/Puerto Rico/8/1934 (H1N1), highpathogenic avian A/Vietnam/03/2004 (H5N1), human A/Hong Kong/8/1968(H3N2), and human A/New York/312/2001 (H1N1). RNA nucleotides numberedin (−)-sense orientation. Sequence alignment of the terminal 5′ regionof PB2 corresponding to the above sequences. Shaded blue box encompassesthe PSL2 RNA secondary structural element. Black dots designatedivergent nucleotide sites.

Mutate-and-Map Strategy Validates PSL2 Structure and Predicts NovelPackaging Mutants

To further test the SHAPE analysis of the PSL2 RNA structure and touncover additional informative mutations needed for in vivo tests,multidimensional chemical mapping (Kladwang and Das, 2010) methods wereapplied to the PSL2 segment. First, mutate-and-map (M²) measurementsconfirmed disruption of the chemical reactivity pattern upon systematicmutation of each stem residue, including changes at nucleotidespreviously found to be critical for PB2 packaging (FIG. 5A, see notedfields) (Marsh et al., 2008; Gog et al., 2007). Automated computationalanalysis based on these M² data recovered the SHAPE-guided PSL2structure with high confidence (FIG. 1C, FIG. 3, FIG. 5B-FIG. 5D),further validating the structural model. Second, as predictive tests,compensatory mutations were designed to restore the base pairs in thewild-type stem-loop structure that were disrupted by the initialpackaging-defective mutations (FIG. 6, panels A-B). Thesemutation-rescue variants indeed restored the PSL2 SHAPE pattern,providing base-pair resolution in vitro validation of the modeledstructure and suggesting sequence variants to test the role of PSL2structure in vivo.

FIG. 5A-FIG. 5D shows 2-Dimensional Mutate-and-Map (M2) analysis of PSL2RNA secondary structure. (FIG. 5A) Systematic single nucleotide mutationand mapping of resulting chemical accessibility reveals interactions inthe three-dimensional structure of the RNA. Chemical accessibilities,plotted in grey scale (black=highest SHAPE reactivity), across 88 singlemutations at single-nucleotide resolutions of PSL2 element from PR8strain PB2. Reactivity peaks (left to right) correspond to nucleotidesfrom the 5′ to 3′ end of the PB2 RNA. Nucleotide sites corresponding toknown packaging mutations (as reported by Marsh et al., 2008) areindicated on right in blue. Red arrows denote prominentpackaging-defective mutant sites predicted by M2 analysis. (FIG. 5B)Strong features of mutate-and-map data isolated by Z-score analysis(number of standard deviations from mean at each residue). Z-scores werecalculated for each nucleotide reactivity by subtracting the averagereactivity of this nucleotide across all mutants and dividing bystandard deviation (output_Zscore_from_rdat in HiTRACE). Squares showsecondary structure model guided by mutate-and-map data. Dark signalshighlight evidence of structured nucleotide pairing. (FIG. 5C) RNAsecondary structure for 5′ packaging signal region (nts 30-93) derivedfrom incorporating Z-scores into the RNAstructure modelling algorithm:bootstrap confidence estimates given as green precentage values.Bootstrap values provide numerically accurate indicators of structuralconfidence. Low bootstrap confidence values suggest existence ofalternative structural models. (FIG. 5D) Bootstrap support values foreach base-pair shown as grayscale shading.

FIG. 6 shows Design of compensatory mutations to previously describedPR8 PB2 mutants. (FIG. 6, panel A) Previously described synonymousmutants (m757, m745, m55c) are mapped onto PSL2 structure. (FIG. 6,panel B) Compensatory mutations (m55c-comp, m745-comp, and m757-comp)were designed at sites predicted to restore wild-type PSL2 structurebased on SHAPE and mutate-and-map chemical analyses. Black boxednucleotides denote site of compensatory mutation. (−)-sense vRNAorientation is shown. For mutations where a non-synonymous change wasrequired to restore the structure, the alteration in encoded proteinsequence is indicated.

To test whether the PSL2 stem-loop structure observed in solution wasrelevant to virus packaging in the cellular milieu, the same ninesynonymous mutations reported by Gog et al., 2007 and Marsh et al., 2008(FIG. 1A-FIG. 1B, FIG. 7 panel A) as well as four new synonymousmutations characterized by M² analysis (FIG. 7, panel B) were clonedinto pDZ plasmids containing the PR8 PB2 gene (Marsh et al., 2008; Lianget al., 2008; Gog et al., 2007) (FIG. 8). The packaging efficiencies ofthe 9 previously known mutants now in the PR8 background were comparableto those originally described in the WSN33 virus¹⁵ (FIG. 7, panel C). Ofthese, mutants m55c, m757, m745, and m744b, were predicted to show themost significant impairment based on their location within PSL2′s stemregions (FIG. 1C, FIG. 3A-FIG. 3F, FIG. 7). In contrast, publishedmutations that have no effect on PB2 packaging (e.g. m731) mapped to theunstructured apical loop or fell outside of PSL2 and did not alter itsstructural integrity (FIG. 9A) (Marsh et al., 2008). The three novelsynonymous mutants (m74-1, m74-2, and m68) identified by M²-analysis ashaving a significant effect on in vitro PSL2 structure (FIG. 5A) showedsignificant loss in PB2 packaging, whereas mutation sites that resultedin negligible change in SHAPE reactivity compared to the wild-type PSL2structure, gave wild-type-like packaging efficiency levels (e.g., m56)(FIG. 7, panel D).

FIG. 7 shows synonymous mutation of single highly conserved codons ofthe PR8 PB2 vRNA. (FIG. 7, panel A) Previously published synonymousmutations implicated in PB2 packaging. Upper line is the parental PR8vRNA sequence ((+)-sense orientation), and the mutated singlenucleotides are bolded in red on the line below. Numbering andnomenclature of introduced mutations are based on the reports by Marshet al., 2008 and Gog et al., 2007. Yellow highlighted region indicatessequence containing PSL2 structure. (FIG. 7, panel B) Design of primersequences for cloning of synonymous mutations identifed from M2-analysis(see Supplemental FIG. 4a ) into pDZ plasmids. Sequences are in(+)-sense orientation. Highlighted nucleotides=mutation site. (FIG. 7,panels C-D) Packaging efficiencies representing the percentage of mutantPB2 packaging relative to parental wild-type PB2 for (FIG. 7, panel C)Previously published synonymous mutants, and (FIG. 7, panel D)M2-analysis identified synonymous mutants. Results from two independentexperiments, assays performed in triplicate (n=6). Error barsrepresent±SD.

FIG. 8 shows PB2 Packaging mutant nomenclature and corresponding sitesof mutation. Mutation nomenclature chart indicating names and site(s) ofmutation from: 1) Previously published synonymous mutations implicatedin PB2 packaging (shown in blue), based on the reports by Marsh et al.,2008 and Gog et al., 2007; and 2) PSL2 structure designed single mutants(shown in black) and double-compensatory mutants (shown in red).Numbering and nomenclature of introduced mutations are based on thegenomic, (−)-sense vRNA. Instances where mutation results in change inprotein coding are indicated by synonymous (SYN) or non-synonymous(Non-SYN) fields.

FIG. 9A-FIG. 9C shows effect of synonymous mutation on PSL2 structure.Left: Predicted RNA secondary structure of PB2 packaging mutantsdetermined by sf-SHAPE analysis on full-length (−)-sense PB2 vRNA fromPR8 strain. For clarity only, the wild-type structure is shown in upperrighthand corner box. Right: SHAPE reactivity graph shown as change ofmutant reactivity over wild-type. Energy values and percent packagingefficiencies indicated below figure headings. Mutants: (FIG. 9A) m731.(FIG. 9B) m751. (FIG. 9C) m748. Percent packaging effiencies of PB2incorporation for each of the previously described mutants arehighlighted in blue.

The compensatory mutations rescued not only the viral packaging forsegment PB2 (FIG. 10, panels A-C, FIG. 6, panels A-B), but also othersegments previously reported to be affected by the deleteriousmutations, consistent with the proposed hierarchal role of PB2 in IAVpackaging (Muramoto et al., 2006; Gao et al., 2012; Marsh et al., 2008)(FIG. 10, panels D-F). In addition to recovering PB2 packaging, thecompensatory mutations gave complete or near-complete rescue of theviral titer loss caused by the defective mutations (FIG. 10, panelsG-I). Some non-synonymous compensatory mutations were able to restorePB2 packaging better than others (m745-comp and m55c-comp, compared tom757-comp) (FIG. 10, panels A-C), possibly reflecting incompleterestoration of PB2 protein function through exogenous addition. Suchexogenous addition was necessitated because some non-synonymousmutations affected both PSL2 structure and protein sequence. The mostincisive test of PSL2 structure came from packaging experiments that didnot require supplemental wild-type PB2 protein addition. Based oncomputational enumeration and multidimensional mutation-rescueexperiments (Tian et al., 2014), a single mutation-rescue pair ofsubstitutions was discovered that were both synonymous, obviatingwild-type PB2 protein addition (m52/m65, FIG. 11, panels A-B, FIG. 12,panel s, FIG. 13). Making each mutation alone (m52 and m65) resulted inpackaging efficiencies below 4% PB2 incorporation and titer lossexceeding 4 log₁₀—an extreme impairment beyond what has been previouslyreported (i.e. 2 log₁₀) for packaging-defective viruses (FIG. 11, panelC-D, FIG. 7, panel c, FIG. 10). When introduced together into a doublymutated m52/65-comp strain that restored PSL2 structure, albeit with analtered sequence, the compensatory mutations restored both packagingefficiency and viral titer to wild-type levels.

FIG. 10, panels A-I, shows effect of compensatory mutations in PR8 PB2packaging-defective mutants on viral packaging and titer. (FIG. 10,panels A-C) Packaging efficiencies of packaging-defective andcompensatory mutant PB2 vRNAs. For compensatory mutations where anon-synonymous change was required, a wild-type PB2 protein expressionplasmid was co-transfected during virus rescue. pWT=expression plasmidencoding for wild-type PR8 PB2 protein. Values given as percentage ofPB2 vRNA packaging in comparison to wt parental PR8 virus. Results fromtwo independent experiments, assays performed in triplicate (n=6). (FIG.panels D-F) Packaging efficiencies of packaging-defective andcompensatory mutant viruses and their effect on the packaging of otherinteracting segments, PB1, PA, NP, and MX. Assays performed intriplicate (n=6). (FIG. 10, panels G-I) Viral titer by plaque assay.Results in PFU/mL, assays performed in triplicate.

FIG. 11 shows multidimensional chemical mapping reveals novel PB2packaging-defective and compensatory mutant partners. (FIG. 11, panel A)Electropherogram result from systematic single nucleotide mutationmapping with rescue (Mutate-Map-Rescue) analysis of individual andcompensatory double mutations to test base-pairings from 1-D-data-guidedmodels and to identify predicted successful synonymous PSL2-defectiveand compensatory mutant pairs. Chemical accessibilities, plotted in greyscale (black=highest SHAPE reactivity), across 88 single mutations atsingle-nucleotide resolutions of PSL2 element from PR8 strain PB2.Reactivity peaks (left to right) correspond to nucleotides from the 5′to 3′ end of the PB2 RNA. See FIG. 12 for complete list of Mutate-Rescuepairs. (FIG. 11, panel B) Mutational design of single mutants m52 (G52U)and m65 (C65A), and the double m52/65 rescue pair on the PSL2 structure.(FIG. 11, panel C) Packaging efficiency of the synonymous single anddouble mutant mutate-and-rescue pair. Values given as percentage of PB2vRNA packaging in comparison to wt parental PR8 virus. Results from twoindependent experiments, assays performed in triplicate (n=6). (FIG. 11,panel D) Viral titer in PFU/mL, results in triplicate. Error barsrepresent±SD.

FIG. 12 panels a-t, shows 2-Dimensional Mutate-Map-Rescue (M2R)analysis. Mutation/Rescue results validate PSL2 RNA secondary structure.Electropherograms of SHAPE analysis with compensatory double mutationsto test base-pairings from 1-D-data-guided models and to identifysuccessful PSL2-defective and compensatory mutant pairs. Chemicalaccessibilities, plotted in grey scale (black=highest SHAPE reactivity),across 88 single mutations at single-nucleotide resolutions of PSL2element from PR8 strain PB2. For each tested pairing, a “quartet” ofwild-type, single mutant 1, single mutant 2, and compensatory doublemutant are grouped for comparison. (FIG. 12, panels a-r) Non-synonymousmutate-and-rescue pairs. All non-boxed electropherograms are pairingsfor which disruption and/or rescue were not observed. Blue boxesindicate successful defective and rescue mutations. (FIG. 12, panel s)Double synonymous mutate-and-rescue pair. Green box=successfulsynonymous defective and rescue pair. (FIG. 12, panel t) Packagingefficiency for non-synonymous mutate-and-rescue pairs. Values given aspercentage of PB2 vRNA packaging in comparison to wt parental PR8 virus.Results from two independent experiments, assays performed in triplicate(n=6). Error bars represent±S.D.

FIG. 13 shows design of primer sequences for 2-DimensionalMutate-Map-Rescue (M2R) mutants. SEQ ID NOs: (28-43) top to bottom.Primer sequences used for QuickChange mutational cloning of M2R mutantsinto pDZ plasmids. Sequences are in (+)-sense orientation. Left fielddennotes synonymous (Syn.) or non-synonymous (Non-syn.) change.Highlighted nucleotides=mutation site. Boxed mutant primer set indicatesdouble synonymous mutant partners, m52 and m65.

To test the relevance of the PSL2 structure in an in vivo model, 6-8week old BALB/C mice were intranasally inoculated with 1000 PFU ofwild-type PR8 viruses or of strains harboring mutations predicted todisrupt or restore PSL2 structure. Mice infected with thePSL2-disrupting mutations—m745 mutant strain (20% packaging efficiency)or the severely packaging-defective single mutant virus, m52 (<4%packaging efficiency)—showed reduced or no clinical signs of illness,respectively, either in weight loss or survival as compared to the PBScontrol (FIG. 14, panels A-B). Remarkably, inclusion of compensatorymutations that restore PSL2 structure rescued viral pathogenicity:animals infected with m52/65-comp and m745-comp, displayed comparablemortality profiles and survival curves as mice infected with wild-typePR8 (FIG. 14, panels A-B). Consistent with APLAC guidelines, all micewere humanely sacrificed upon reaching greater than 20% weight loss.

FIG. 14 panels A-B shows packaging-defective viruses are attenuated invivo. Percent weight loss and survival of mice infected with singlePSL2-disrupting and compensatory, PSL2-restoring double-mutant viruses.6-8 week old BALB/C female mice were intranasally infected with 1000PFUof PR8 wild-type (wt) virus, packaging defective single mutant viruses,m52 and m745, compensatory double mutant viruses, m52/65 and m745-comp,or PBS control. Mice were monitored daily for percent day 0 weight lossand percent survival. Results as an average of two independentexperiments, 6 mice per condition. (FIG. 14, panel A) Percent weightloss. (FIG. 14, panel B) Kaplan-Meir survival plot of the individualcohorts depicted in (FIG. 14, panel A).

Example 2 Therapeutic Design and Targeting of PSL2 Structure InhibitsIAV Infection In Vitro and In Vivo

To explore the therapeutic potential of targeting PSL2-mediated viralpackaging, nine locked nucleic acids (LNAs) containing phosphorothioateinternucleoside linkages (Vester and Wengel, 2004) were designed againstkey residues predicted to disrupt the overall RNA secondary structure ofthe element and thereby inhibit viral production (FIG. 15, panel A). Twoof the designed LNAs, LNA8 and LNA9, are identical in sequence to LNA6and LNA7, respectively, but possess 6-7 unmodified (non-locked) DNAnucleotides optimized for RNase-H activation. First, to assess theimpact that LNA binding has on PSL2 RNA secondary structure, toeprintingand SHAPE chemical mapping were performed on PB2 vRNA in the presence ofthe LNAs. In corroboration with the antiviral assay results, sequencesencoded in LNAs 6-9 exhibited the greatest ability to bind and disruptthe wild-type PSL2 structure (FIG. 16).

FIG. 15, panels A-D shows Locked Nucleic Acids targeting PSL2 RNAstructure display potent antiviral activity in vitro and in vivo. (FIG.15, panel A) Location of complementary Locked Nucleic Acids (LNAs)designed against different regions of the PSL2 structure. (FIG. 15,panel B) To screen the LNAs for antiviral activity, MDCK cells werepretreated with 100 nM of each designated LNA by Lipofectaminetransfection for 1 hr prior to infection with 0.01 MOI of either PR8(H1N1) virus or A/Hong Kong/8/68 (H3N2) virus. 48 hours post-infection,supernatant was collected and viral titers determined by plaque assay.Results from 2 independent experiments, assays performed in triplicate(n=6). (FIG. 15, panel C) Time course of pre-treatment (RX) versuspost-infection treatment with LNA9 at titrating concentrations (100 nM,10 nM, 1 nM). WT+Lipo=infection with Lipofectamine control.Pretreatment: confluent MDCK cells in 6-well plates were treated withLNA9 either 2 hours or 4 hours prior to infection. Treated supernatantwas removed at the indicated time point and cells were infected with0.01 MOI of wt PR8 virus for one hour. For post-infection treatment:MDCK cells were infected with 0.01 MOI of PR8 virus for one hour, afterwhich the supernatant was replaced and the cells were treated with LNA9at either 2 or 4 hours post-infection. Supernatant was collected 48 hrslater, and viral titers determined by plaque assay in triplicate. FIG.15, panel d shows the effects of intranasal LNA treatment on survival ofvirus-infected mice. Mice were intranasally administered 20 ug of LNA9,scrambled LNA, or PBS (uninfected control) at 12 hours prior toinfection with PR8 virus. All mice received two additional treatments at8 hpi and 36 hpi (n=7 mice per condition).

FIG. 16A-FIG. 16B shows SHAPE analysis on LNA-RNA binding. (FIG. 16A)Electrophoretic profile of SHAPE analysis performed on LNAs 1, 2, 4, 5,6/8, and 7/9 (100 nM) in the presence of PR8 PB2 vRNA. S1, a smallmolecule that interacts with the Hepatitis C virus IRES RNA was added asa control for RNA binding. Columns on the left, unlabeled reactionwithout SHAPE reagent, 1M7. Right: with labeling reagent. (FIG. 16B)Electrophoretic profile of SHAPE performed on LNA-vRNA combination intitrating concentrations of LNA. For each LNA, the left set of columnsis without labeling reagent.

Then, in a first pilot experiment to determine the antiviral potentialof LNA-mediated targeting of PSL2 across two different IAV subtypes,MDCK cells were pre-treated with 100 nM of each LNA and delivered byLipofectamine™ transfection for 1 hour prior to infection with 0.01 MOIof either the wild-type PR8 (H1N1) virus or tissue-culture adaptedA/Hong Kong/8/68 (HK68) (H3N2) virus. Forty-eight hours post-infection,the supernatants were collected and viral production was measured byplaque assay (FIG. 15, panel B). LNAs directed against only the top loopof PSL2 (LNA1, LNA4) had little to no effect on viral titer. Similarly,LNAs 3 and 5, which target the 3′ base of PSL2, also failed to inhibitviral production. In contrast, nucleotide coverage of both the top loopand middle bulge by LNA6 resulted in greater than 2 log₁₀ titer deficitsfor PR8 (FIG. 15, panels A-B). LNA8, the RNase-H activated copy of LNA6,produced even greater antiviral activity against both viruses of up to 3logs. Most strikingly, LNA9, the RNase-H activated copy of LNA7,possessed the strongest antiviral capacity, dropping viral production bynearly 5 logs and 4 logs against PR8 and HK68, respectively.

Having identified the optimal candidate LNAs, the treatment time-courseand concentration parameters of LNA9's antiviral activity were furtherinvestigated. MDCK cells were treated with one dose of 10-fold dilutionsof LNA9 at either 2 or 4 hours pre-infection or, alternatively, 2 or 4hours post-infection with 0.01 MOI of wild-type PR8 virus. Cellspre-treated with the LNA had the most potent antiviral response (greaterthan 4 logs), and displayed strong viral inhibition (greater than 2logs) even at the lowest dilution (1 nM) (FIG. 15, panel C). There was atrend towards decreasing antiviral activity as the time post-infectiontreatment increased, but even at the latest tested time point ofaddition, greater than 3 logs suppression of viral titer was achieved.

Example 3 In Vivo Efficacy Experiment: Extended Single-Dose Prophylaxis

Balb/C female mice (5 mice/group) were pre-treated intranasally with asingle dose of 20 ug LNA9 either 3 days before infection (Day −3) or 1day before infection (Day −1) with a lethal dose of wild-type PR8 virus.Mice were monitored daily for weight loss, clinical score, and survival.FIG. 17, panel A shows the percent survival of mice over time. FIG. 17,panel B shoes the percent weight loss over time after administration.

A single administration of 20 ug LNA9 three days prior to infectioncompletely protected mice from fatal influenza disease. Non-treatedcontrol mice were humanely sacrificed when they lost greater than 25%weight loss, at an average of 5.5 days. In contrast, the pretreatmentgroup showed minimal weight loss, little-to-no clinical signs ofdisease, and fully recovered to pre-infection weight.

This result demonstrates that a single, inhalable dose of LNA9administered days before infection can provide lasting protectionagainst fatal disease, and suggests the subject compounds can find usein prophylaxis treatment during influenza outbreaks and pandemics.

Example 4 Susceptibility of Influenza Viruses to Oseltamivir and LNA9,After Serial Passaging in the Presence of Drug: Drug SelectionExperiment

Oseltamivir (Tamiflu) is the most widely used and stockpiledneuraminidase inhibitor (NAI) on the market. Like all NAIs, oseltamivirrequires a conformational rearrangement in the viral neuraminidase (NA)protein to accommodate the drug. Any mutations in the NA protein thataffect this rearrangement reduce the binding affinity of oseltamivir,thus reducing drug efficacy. Notably, the H274Y mutant (also known asthe H275Y mutation depending on nomenclature) is most commonlyassociated with oseltamivir resistance. The rapid selection of the H274Ymutation in an immunocompromised patient can lead to clinical failure ofthe last-resort NAI drug, peramivir, suggesting that the selection formulti-drug resistant viruses in immunocompromised hosts may be morecommon than previously believed. This, together with the recent spreadof oseltamivir-resistant and NAI-resistant viruses in circulation,indicates the need to reevaluate usage of NAIs in general. Thedevelopment of new classes of antivirals is imperative in order toreduce the adverse impact current and future influenza pandemics canhave on human health.

The sequence region in segment PB2 that contains the PSL2 stemloop ishighly conserved across IAV subtypes, strains and isolates from a widerange of host-species, and likely reflects a strict biologic requirementfor its preservation. SHAPE analysis of this region confirmedmaintenance of the PSL2 structure between seasonal as well as pandemicviruses of different subtypes and host origins, strongly suggesting thatthis structural element could be a novel pan-genotypic therapeutictarget (and hence LNA9 has broad spectrum potential against IAVisolates). Additionally, since the subject LNAs are directed against ahighly conserved viral genomic RNA target that clearly has strongconstraints on its ability to mutate, the subject LNAs targeting PSL2are expected to have a higher barrier to the development of resistancecompared to NAIs.

The susceptibility of influenza viruses to LNA9 versus oseltamivir afterserial passages under drug pressure was investigated. Oseltamivir had astarting IC₅₀ of 41 nM against PR8 at passage 1 of drug treatment, asdetermined by a plaque reduction assay. After only six virus passageswith increasing amounts of drug, the IC₅₀ of oseltamivir leapt to 50uM-a 1000× increase fold. See FIG. 18, panels A-D. In comparison, after10 passages of virus in the presence of LNA9, the IC₅₀ held stable from18 to 16 pM. FIG. 19, panels A and B.

LNA9 can also be used to treat drug resistant viruses. A drug-resistantmutant of A/WSN/33 (H1N1) virus was generated using a reverse geneticvirus rescue system that mutated the NA gene to contain the H274Yresistance mutation. Against this virus, oseltamivir has an IC₅₀ of 53uM. Importantly, LNA9 maintained its potency and efficacy against theWSN H274Y virus with picomolar activity. FIG. 19, panel C. This resultis strong evidence for the therapeutic treatment of NAI-resistantviruses with PSL2-targeting LNAs. This result also highlights theactivity of LNA9 against different IAV isolates.

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Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the embodimentsshown and described herein. Rather, the scope and spirit of presentinvention is embodied by the appended embodiments.

Notwithstanding the appended claims, the disclosure set forth herein isalso described by the following clauses:

Clause 1. An oligonucleotide compound, comprising an oligonucleotidesequence complementary to a PB2 vRNA region, wherein the regioncomprises nucleotides 34-87 in the (−)-sense notation of the 5′ terminalcoding region of the PB2 vRNA, or a salt thereof.

Clause 2. The compound of clause 1, comprising an oligonucleotidesequence comprising at least 8 nucleoside subunits complementary to theregion of PB2 vRNA.

Clause 3. The compound of any one of clauses 1-2, wherein theoligonucleotide is complementary to a region of the Packaging Stem-Loop2 (PSL2) motif of the region of PB2 vRNA.

Clause 4. The compound of any one of clauses 1-3, wherein theoligonucleotide comprises an internucleoside linkage selected from:phosphorothioate, phosphorodithioate, phosphoramidate andthiophosphoramidate linkages.

Clause 5. The compound of any one of clauses 1-4, wherein all of theinternucleoside linkages of the oligonucleotide are selected from:phosphorothioate, phosphorodithioate, phosphoramidate,thiophosphoramidate and phosphodiester linkages.

Clause 6. The compound of any one of clauses 1-5, wherein theoligonucleotide comprises a locked nucleic acid (LNA) nucleotide.

Clause 7. The compound of any one of clauses 1-6, wherein theoligonucleotide comprises a sequence selected from:

(SEQ ID NO: 45) 5′ ACCAAAAGAAT 3′; (SEQ ID NO: 46) 5′ TGGCCATCAAT 3′;(SEQ ID NO: 47) 5′ TAGCATACTTA 3′; (SEQ ID NO: 48) 5′ CCAAAAGA 3′;(SEQ ID NO: 49) 5′ CATACTTA 3′; (SEQ ID NO: 50) 5′ CAGACACGACCAAAA 3′;(SEQ ID NO: 51) 5′ TACTTACTGACAGCC 3′; (SEQ ID NO: 52) 5′AGACACGACCAAAAG 3′; (SEQ ID NO: 53) 5′ ACCAAAAGAAT 3′; (SEQ ID NO: 54)5′ TGGCCATCAAT 3′; (SEQ ID NO: 55) 5′ TAGCATACTTA 3′; (SEQ ID NO: 56) 5′CGACCAAAAGAATTC 3′; (SEQ ID NO: 57) 5′ CGACCAAAAGAATTC 3′;(SEQ ID NO: 58) 5′ GATGGCCATCAATTA 3′; (SEQ ID NO: 59) 5′GATGGCCATCAATTA 3′; (SEQ ID NO: 60) 5′ TCTAGCATACTTACT 3′;(SEQ ID NO: 61) 5′ TCTAGCATACTTACT 3′; (SEQ ID NO: 62) 5′GAATTCGGATGGCCA 3′; (SEQ ID NO: 63) 5′ GGCCATCAATTAGTG 3′;(SEQ ID NO: 64) 5′ TTCGGATGGCCATCA 3′; (SEQ ID NO: 65) 5′AGCCAGACAGCGA 3′; and (SEQ ID NO: 66) 5′ GACAGCCAGACAGCA 3′.

Clause 8. The compound of clause 7, wherein all the nucleotides of theoligonucleotide are locked nucleic acid (LNA) nucleotides.

Clause 9. The compound of clause 7, wherein the oligonucleotidecomprises a sequence selected from:

(SEQ ID NO: 67) LNA 1: 5′ AccAaaAGaaT 3′; (SEQ ID NO: 68) LNA 2: 5′TggCcATcaaT 3′; (SEQ ID NO: 69) LNA 3: 5′ TagCAtActtA 3′;(SEQ ID NO: 70) LNA 4: 5′ CCAAAAGA 3′; (SEQ ID NO: 71) LNA 5: 5′CATACTTA 3′; (SEQ ID NO: 72) LNA 6: 5′ CagaCaCGaCCaaAA 3′;(SEQ ID NO: 73) LNA 7: 5′ TAcTtaCTgaCagCC 3′; (SEQ ID NO: 74) LNA 8: 5′AGACacgaccaAAAG 3′; (SEQ ID NO: 75) LNA 9: 5′ TACTtactgacaGCC 3′;(SEQ ID NO: 76) LNA9.2: 5′ TACttactgacAGCC 3′; (SEQ ID NO: 77) LNA10:5′ACCaaaagAAT 3′; (SEQ ID NO: 78) LNA11: 5′ TGGccatcAAT 3′;(SEQ ID NO: 79) LNA12: 5′TAGcatacTTA 3′; (SEQ ID NO: 80) LNA13:5′CgacCAaaAGaattC 3′; (SEQ ID NO: 81) LNA14: 5′CGACcaaaagaATTC 3′;(SEQ ID NO: 82) LNA15: 5′GaTGgCcATcaAttA 3′; (SEQ ID NO: 83) LNA16:5′GATGgccatcaATTA 3′; (SEQ ID NO: 84) LNA17: 5′TcTAgCaTActTacT 3′;(SEQ ID NO: 85) LNA18: 5′TCTAgcatactTACT 3′; (SEQ ID NO: 86) LNA19:5′GAAttcggatgGCCA 3′; (SEQ ID NO: 87) LNA20: 5′GGCCatcaattaGTG 3′;(SEQ ID NO: 88) LNA21: 5′TTCGgatggccaTCA 3′; (SEQ ID NO: 89) LNA22:5′AGCCagacagCGA 3′; (SEQ ID NO: 90) LNA23: 5′GACAgccagacaGCA 3′;(SEQ ID NO: 91) LNA 9.G74C: 5′TACTtactgacaGTC 3′; and (SEQ ID NO: 92)LNA 9.T80C: 5′TACTtaccgacaGCC 3′;

wherein capitalized letters denote LNA nucleotides and lowercase lettersdenote DNA nucleotides.

Clause 10. The compound of any one of clauses 1-9, wherein theoligonucleotide comprises at least 5 deoxyribonucleotide units and iscapable of recruiting an RNase.

Clause 11. The compound of any one of clauses 1-10, wherein binding ofthe compound to the region of PB2 vRNA disrupts the overall secondaryRNA structure of the PB2 vRNA.

Clause 12. The compound of any one of clauses 1-11, wherein the compoundis an oligonucleotide conjugate having enhanced cellular uptake.

Clause 13. The compound of clause 12, wherein the compound is anoligonucleotide-lipid conjugate.

Clause 14. The compound of any one of clauses 1-12, wherein the compoundis an oligonucleotide conjugate with a cell-specific protein.

Clause 15. A method of inhibiting influenza A virus in a cell, themethod comprising: contacting a sample comprising viral RNA (vRNA)having a PSL2 motif with an effective amount of an agent thatspecifically binds the PSL2 motif to inhibit the influenza A virus.

Clause 16. The method of clause 15, wherein the agent is anoligonucleotide compound comprising at least 8 nucleoside subunitscomplementary to a PSL2 motif of the vRNA, or a salt thereof.

Clause 17. The method of clause 15 or 16, wherein the agent is anoligonucleotide compound according to one of clauses 1-14.

Clause 18. The method of any one of clauses 15-17, wherein the vRNA inthe sample is a PB2 vRNA.

Clause 19. The method of any one of clauses 15-18, wherein contactingthe sample with an agent results in at least 2 log₁₀ titer deficits ofthe virus.

Clause 20. The method of any one of clauses 15-19, wherein the agentdisrupts the overall structure of the PSL2 motif of the vRNA.

Clause 21. The method of any one of clauses 15-19, wherein the vRNA isisolated from a virion or a cell.

Clause 22. The method of any one of clauses 15-19, wherein the vRNA iscomprised in a virion or an infected cell.

Clause 23. The method of any one of clauses 15-22, wherein the sample isin vitro.

Clause 24. The method of any one of clauses 15-19 or 22, wherein thesample is in vivo.

Clause 25. A method of treating or preventing influenza A virusinfection in a subject, the method comprising: administering to asubject in need thereof a pharmaceutical composition comprising aneffective amount of an active agent that specifically binds to a PSL2motif of a viral RNA (vRNA).

Clause 26. The method of clause 25, wherein the vRNA is a PB2 vRNA.

Clause 27. The method of any one of clauses 25-26, wherein the activeagent is a compound comprising an oligonucleotide sequence comprising atleast 8 nucleoside subunits complementary to the region of PB2 vRNA.

Clause 28. The method of any one of clauses 25-27, wherein the agent isan oligonucleotide compound according to one of clauses 1-14.

Clause 29. The method of any one of clauses 25-28, wherein the subjectis at risk of influenza A virus infection and the administering of theoligonucleotide compound protects the subject against infection for 1week or more (e.g., 2 weeks or more, 3 weeks or more, 1 month or more, 2months or more, 3 months or more, etc).

Clause 30 The method of clause 29, wherein the administering comprisesweekly, biweekly, or monthly administration of an effective dose of theoligonucleotide compound.

Clause 31. The method of any one of clauses 25-30, wherein theadministering results in at least 2 log_(in) titer deficits of the virusin a sample of the subject.

Clause 32. The method of any one of clauses 25-30, wherein the activeagent is an oligonucleotide conjugate having enhanced cellular uptake.

Clause 33. The method of any one of clauses 25-31, wherein the activeagent is an oligonucleotide conjugate with a cell-specific protein.

Clause 34. The method of any one of clauses 25-31, wherein thepharmaceutical composition comprises an enhancer of cellular uptake.

Clause 35. The method of any one of clauses 25-31, wherein thepharmaceutical composition further comprises an additional active agentselected from a second oligonucleotide active agent and an antiviraldrug.

Clause 36. The method of any one of clauses 25-31, wherein the activeagent is an siRNA, an shRNA, an antisense RNA, or an antisense DNA.

Clause 37. The method of any one of clauses 25-31, wherein the subjectis at risk of influenza A virus infection, and the method preventsinfection.

Clause 38. The method of any one of clauses 25-31, wherein the subjectis diagnosed with or suspect of having an influenza A virus infection,and the method treats infection.

Clause 39. A method for screening a candidate agent for the ability toinhibit influenza A virus in a cell, the method comprising:

contacting a sample comprising viral RNA (vRNA) comprising a PSL2 motifwith a candidate agent; and

determining whether the candidate agent specifically binds to the PSL2motif;

wherein an agent that specifically binds to the PSL2 motif will inhibitinfluenza A virus in a cell.

Clause 40. The method of clause 39, wherein the candidate agent isselected from: a small molecule, a nucleic acid and a polypeptide.

Clause 41. The method of clause 40, wherein the determining stepcomprises detecting a cellular parameter, wherein a change in theparameter in the cell as compared to in a cell not contacted withcandidate agent indicates that the candidate agent specifically bindsthe PSL2 motif.

Clause 42. The method of any one of clauses 39-41, wherein an agent thatspecifically binds to the PSL2 motif will treat the subject having theinfluenza A virus infection.

What is claimed is:
 1. An oligonucleotide compound or salt thereof,comprising an oligonucleotide sequence comprising at least 8 nucleosidesubunits complementary to a region of a Packaging Stein-Loop 2 (PSL2)motif of a PB2 viral RNA (vRNA) or mutant thereof, wherein theoligonucleotide compound inhibits virus production and theoligonucleotide sequence is selected from: (SEQ ID NO:45) 5'ACCAAAAGAAT 3';  (SEQ ID NO:50) 5' CAGACACGACCAAAA 3';  (SEQ ID NO:51)5' TACTTACTGACAGCC 3';  (SEQ ID NO:52) 5' AGACACGACCAAAAG 3'; (SEQ ID NO:56) 5' CGACCAAAAGAATTC 3';  (SEQ ID NO:58) 5'GATGGCCATCAATTA 3';  (SEQ ID NO:60) 5' TCTAGCATACTTACT 3'; (SEQ ID NO:63) 5' GGCCATCAATTAGTG 3';  (SEQ ID NO:64) 5'TTCGGATGGCCATCA 3';  (SEQ ID NO:66) 5' GACAGCCAGACAGCA 3'; (SEQ ID NO:91) 5' TACTTACTGACAGTC 3';  (SEQ ID NO:92) 5'TACTTACCGACAGCC 3'; and  (SEQ ID NO:93)  5' GGATTTCGGATGGCCA 3'.


2. The compound of claim 1, wherein the oligonucleotide comprises aninternucleoside linkage selected from: phosphorothioate,phosphorodithioate, phosphoramidate and thiophosphoramidate linkages. 3.The compound of claim 1, wherein the oligonucleotide comprises a lockednucleic acid (LNA) nucleotide.
 4. The compound of claim 1, wherein theoligonucleotide comprises at least 5 deoxyribonucleotide units and iscapable of recruiting an RNase.
 5. The compound of claim 3, wherein theoligonucleotide sequence comprises an LNA-containing sequence selectedfrom: (SEQ ID NO: 67) LNA 1: 5′ AccAaaAGaaT 3′; (SEQ ID NO: 68) LNA 2:5′ TggCcATcaaT 3′; (SEQ ID NO: 69) LNA 3: 5′ TagCAtActtA 3′;(SEQ ID NO: 70) LNA 4: 5′ CCAAAAGA 3′; (SEQ ID NO: 71) LNA 5: 5′CATACTTA 3′; (SEQ ID NO: 72) LNA 6: 5′ CagaCaCGaCCaaAA 3′;(SEQ ID NO: 73) LNA 7: 5′ TAcTtaCTgaCagCC 3′; (SEQ ID NO: 74) LNA 8: 5′AGACacgaccaAAAG 3′; (SEQ ID NO: 75) LNA 9: 5′ TACTtactgacaGCC 3′;(SEQ ID NO: 76) LNA9.2: 5′ TACttactgacAGCC 3′; (SEQ ID NO: 77) LNA10:5′ACCaaaagAAT 3′; (SEQ ID NO: 78) LNA11: 5′ TGGccatcAAT 3′;(SEQ ID NO: 79) LNA12: 5′TAGcatacTTA 3′; (SEQ ID NO: 80) LNA13:5′CgacCAaaAGaattC 3′; (SEQ ID NO: 81) LNA14: 5′CGACcaaaagaATTC 3′;(SEQ ID NO: 82) LNA15: 5′GaTGgCcATcaAttA 3′; (SEQ ID NO: 83) LNA16:5′GATGgccatcaATTA 3′; (SEQ ID NO: 84) LNA17: 5′TcTAgCaTActTacT 3′;(SEQ ID NO: 85) LNA18: 5′TCTAgcatactTACT 3′; (SEQ ID NO: 86) LNA19:5′GAAttcggatgGCCA 3′; (SEQ ID NO: 87) LNA20: 5′GGCCatcaattaGTG 3′;(SEQ ID NO: 88) LNA21: 5′TTCGgatggccaTCA 3′; (SEQ ID NO: 89) LNA22:5′AGCCagacagCGA 3′; (SEQ ID NO: 90) LNA23: 5′GACAgccagacaGCA 3′;(SEQ ID NO: 91) LNA 9.G74C: 5′TACTtactgacaGTC 3′; and (SEQ ID NO: 92)LNA 9.T80C: 5′TACTtaccgacaGCC 3′;

wherein capitalized letters denote LNA nucleotides and lowercase lettersdenote DNA nucleotides.
 6. A method of inhibiting influenza A virus in acell, the method comprising: contacting a sample comprising viral RNA(vRNA) having a PSL2 motif with an effective amount of theoligonucleotide compound according to claim
 1. 7. The method of claim 6,wherein contacting the sample with an agent results in at least 2 log₁₀titer deficits of the virus or the agent disrupts the overall structureof the PSL2 motif of the vRNA.
 8. The method of claim 6, wherein thevRNA is present in a virion or a cell.
 9. A method of treating orpreventing influenza A virus infection in a subject, the methodcomprising: administering to a subject in need thereof a pharmaceuticalcomposition comprising an effective amount of the oligonucleotidecompound according to claim
 1. 10. The method of claim 9, wherein thesubject is at risk of influenza A virus infection and the administeringof the oligonucleotide compound protects the subject against infectionfor 1 week or more.
 11. The method of claim 10, wherein theadministering comprises weekly, biweekly, or monthly administration ofan effective dose of the oligonucleotide compound.
 12. The method ofclaim 9, wherein the pharmaceutical composition further comprises anadditional active agent selected from a second oligonucleotide activeagent and an antiviral drug.
 13. The method of claim 9, wherein thesubject is diagnosed with or suspected of having an influenza A virusinfection.
 14. The compound of claim 1, wherein the oligonucleotidesequence is selected from: (SEQ ID NO:50) 5' CAGACACGACCAAAA 3'; (SEQ ID NO:51) 5' TACTTACTGACAGCC 3';  (SEQ ID NO:52) 5'AGACACGACCAAAAG 3';  (SEQ ID NO:56) 5' CGACCAAAAGAATTC 3'; (SEQ ID NO:58) 5' GATGGCCATCAATTA 3';  (SEQ ID NO:60) 5'TCTAGCATACTTACT 3';  (SEQ ID NO:63) 5' GGCCATCAATTAGTG 3'; (SEQ ID NO:64) 5' TTCGGATGGCCATCA 3';  (SEQ ID NO:66) 5'GACAGCCAGACAGCA 3';  (SEQ ID NO:91) 5' TACTTACTGACAGTC 3'; and (SEQ ID NO:92) 5' TACTTACCGACAGCC 3'. 


15. An oligonucleotide compound or salt thereof, comprising anoligonucleotide sequence comprising at least 8 nucleoside subunitscomplementary to a region of a Packaging Stem-Loop 2 (PSL2) motif of aPB2 viral RNA (vRNA) or mutant thereof, wherein the oligonucleotidesequence is selected from: (SEQ ID NO:51) 5' TACTTACTGACAGCC 3'; (SEQ ID NO:58) 5' GATGGCCATCAATTA 3';  (SEQ ID NO:60) 5'TCTAGCATACTTACT 3';  (SEQ ID NO:63) 5' GGCCATCAATTAGTG 3'; (SEQ ID NO:64) 5' TTCGGATGGCCATCA 3';  (SEQ ID NO:66) 5'GACAGCCAGACAGCA 3';  (SEQ ID NO:91) 5' TACTTACTGACAGTC 3'; and (SEQ ID NO:92) 5' TACTTACCGACAGCC 3'. 


16. The method according to claim 6, wherein the oligonucleotidesequence is selected from: (SEQ ID NO:50) 5' CAGACACGACCAAAA 3'; (SEQ ID NO:51) 5' TACTTACTGACAGCC 3';  (SEQ ID NO:52) 5'AGACACGACCAAAAG 3';  (SEQ ID NO:56) 5' CGACCAAAAGAATTC 3'; (SEQ ID NO:58) 5' GATGGCCATCAATTA 3';  (SEQ ID NO:60) 5'TCTAGCATACTTACT 3';  (SEQ ID NO:63) 5' GGCCATCAATTAGTG 3'; (SEQ ID NO:64) 5' TTCGGATGGCCATCA 3';  (SEQ ID NO:66) 5'GACAGCCAGACAGCA 3';  (SEQ ID NO:91) 5' TACTTACTGACAGTC 3'; and (SEQ ID NO:92) 5' TACTTACCGACAGCC 3'. 


17. The method according to claim 16, wherein the oligonucleotidesequence is selected from: (SEQ ID NO:51) 5' TACTTACTGACAGCC 3'; (SEQ ID NO:58) 5' GATGGCCATCAATTA 3';  (SEQ ID NO:60) 5'TCTAGCATACTTACT 3';  (SEQ ID NO:63) 5' GGCCATCAATTAGTG 3'; (SEQ ID NO:64) 5' TTCGGATGGCCATCA 3';  (SEQ ID NO:66) 5'GACAGCCAGACAGCA 3';  (SEQ ID NO:91) 5' TACTTACTGACAGTC 3'; and (SEQ ID NO:92) 5' TACTTACCGACAGCC 3'. 


18. The method according to claim 9, wherein the oligonucleotidesequence is selected from: (SEQ ID NO:50) 5' CAGACACGACCAAAA 3'; (SEQ ID NO:51) 5' TACTTACTGACAGCC 3';  (SEQ ID NO:52) 5'AGACACGACCAAAAG 3';  (SEQ ID NO:56) 5' CGACCAAAAGAATTC 3'; (SEQ ID NO:58) 5' GATGGCCATCAATTA 3';  (SEQ ID NO:60) 5'TCTAGCATACTTACT 3';  (SEQ ID NO:63) 5' GGCCATCAATTAGTG 3'; (SEQ ID NO:64) 5' TTCGGATGGCCATCA 3';  (SEQ ID NO:66) 5'GACAGCCAGACAGCA 3';  (SEQ ID NO:91) 5' TACTTACTGACAGTC 3'; and (SEQ ID NO:92) 5' TACTTACCGACAGCC 3'. 


19. The method according to claim 18, wherein the oligonucleotidesequence is selected from: (SEQ ID NO:51) 5' TACTTACTGACAGCC 3'; (SEQ ID NO:58) 5' GATGGCCATCAATTA 3';  (SEQ ID NO:60) 5'TCTAGCATACTTACT 3';  (SEQ ID NO:63) 5' GGCCATCAATTAGTG 3'; (SEQ ID NO:64) 5' TTCGGATGGCCATCA 3';  (SEQ ID NO:66) 5'GACAGCCAGACAGCA 3';  (SEQ ID NO:91) 5' TACTTACTGACAGTC 3'; and (SEQ ID NO:92) 5' TACTTACCGACAGCC 3'. 